Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility.

BACKGROUND: Lignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing. RESULTS: Arabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes. CONCLUSIONS: Simultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Xylobiose treatment triggers a defense-related response and alters cell wall composition.

Plant cell wall-derived oligosaccharides, i.e., damage-associated molecular patterns (DAMPs), could be generated after pathogen attack or during normal plant development, perceived by cell wall receptors, and can alter immunity and cell wall composition. Therefore, we hypothesised that xylo-oligosaccharides (XOS) could act as an elicitor and trigger immune responses. To test this, we treated Arabidopsis with xylobiose (XB) and investigated different parameters. XB-treatment significantly triggered the generation of reactive oxygen species (ROS), activated MAPK protein phosphorylation, and induced callose deposition. The combination of XB (DAMP) and flg22 a microbe-associated molecular pattern (MAMP) further enhanced ROS response and gene expression of PTI marker genes. RNA sequencing analysis revealed that more genes were differentially regulated after 30 min compared to 24 h XB-treated leaves, which correlated with ROS response. Increased xylosidase activity and soluble xylose level after 30 min and 3 h of XB-treatment were observed which might have weakened the DAMP response. However, an increase in total cell wall sugar and a decrease in uronic acid level was observed at both 30 min and 24 h. Additionally, arabinose, rhamnose, and xylose levels were increased in 30 min, and glucose was increased in 24 h compared to mock-treated leaves. The level of jasmonic acid, abscisic acid, auxin, and cytokinin were also affected after XB treatment. Overall, our data revealed that the shortest XOS can act as a DAMP, which triggers the PTI response and alters cell wall composition and hormone level.

Assessing effectiveness of braille and audio-tactile performance technique in improving oral hygiene status of young adults with visual impairment.

INTRODUCTION: Young adults with Visual impairment (VI) challenge the dentists' skills and knowledge due to a higher risk of developing oral diseases due to difficulty in attaining good oral hygiene (OH). AIMS: To assess the effectiveness of ATP (Audio-Tactile Performance) technique and braille versus braille alone in improving OH status of young adults with VI. METHODS AND MATERIAL: A parallel arm, randomized controlled trial was conducted among 70 young adults with VI. Participants were randomly allocated to either test group (braille combined with ATP) or control group (braille alone). A pre-validated braille questionnaire was used to obtain the baseline data followed by clinical examination. OH status assessment was recorded through Gingival Index (GI) and Plaque Index (PI) proforma followed by a thorough ultrasonic oral prophylaxis. Periodic reinforcement was carried out on the 7th day, after 1 month and 3 months. The outcomes were assessed at the end of 3rd and 6th month. RESULTS: There was an increase in the knowledge scores after 3 and 6 months and attitude scores, GI scores and PI scores after 6 months in the test group compared to the control group and this difference was found to be statistically significant. CONCLUSIONS: The findings of this study showed that a combination of ATP and braille proved to be more effective than braille alone in improving the knowledge and OH status of young adults with VI.

Morpho-biochemical characterization of a RIL population for seed parameters and identification of candidate genes regulating seed size trait in lentil (Lens culinaris Medik.).

The seed size and shape in lentil (Lens culinaris Medik.) are important quality traits as these influences the milled grain yield, cooking time, and market class of the grains. Linkage analysis was done for seed size in a RIL (F5:6) population derived by crossing L830 (20.9 g/1000 seeds) with L4602 (42.13 g/1000 seeds) which consisted of 188 lines (15.0 to 40.5 g/1000 seeds). Parental polymorphism survey using 394 SSRs identified 31 polymorphic primers, which were used for the bulked segregant analysis (BSA). Marker PBALC449 differentiated the parents and small seed size bulk only, whereas large seeded bulk or the individual plants constituting the large-seeded bulk could not be differentiated. Single plant analysis identified only six recombinant and 13 heterozygotes, of 93 small-seeded RILs (<24.0 g/1000 seed). This clearly showed that the small seed size trait is very strongly regulated by the locus near PBLAC449; whereas, large seed size trait seems governed by more than one locus. The PCR amplified products from the PBLAC449 marker (149bp from L4602 and 131bp from L830) were cloned, sequenced and BLAST searched using the lentil reference genome and was found amplified from chromosome 03. Afterward, the nearby region on chromosome 3 was searched, and a few candidate genes like ubiquitin carboxyl-terminal hydrolase, E3 ubiquitin ligase, TIFY-like protein, and hexosyltransferase having a role in seed size determination were identified. Validation study in another RIL mapping population which is differing for seed size, showed a number of SNPs and InDels among these genes when studied using whole genome resequencing (WGRS) approach. Biochemical parameters like cellulose, lignin, and xylose content showed no significant differences between parents and the extreme RILs, at maturity. Various seed morphological traits like area, length, width, compactness, volume, perimeter, etc., when measured using VideometerLab 4.0 showed significant differences for the parents and RILs. The results have ultimately helped in better understanding the region regulating the seed size trait in genomically less explored crops like lentils.

Combined Deltoid and Spring Ligament Reconstruction Using the Quadrangular Construct: Our Experience and Review of the Literature.

Background A combined reconstruction of chronic deltoid and spring ligament insufficiency is uncommon. Our study aims to share our experience in treating post-traumatic, chronic deltoid, and spring ligament insufficiency using the "quadrangular construct" technique. Material and methods Five patients who had post-traumatic combined deltoid and spring ligament insufficiency were included in the study. All patients reported a "giving-way" sensation. Preoperatively, each patient underwent weight-bearing radiographs of the ankle and foot. The talo-first metatarsal angle and hindfoot alignment angle were noted. The superficial deltoid ligament was repaired using a suture anchor augmented with Internal BraceTM (Arthrex, Naples, USA) FiberTape® to form a quadrangular construct that anatomically mimics various components of the deltoid-spring ligament complex. Due to the associated excessive heel valgus, three patients also underwent medial displacement calcaneum osteotomy. Additionally, one patient required lateral ligament repair, and another patient required syndesmotic stabilization. The American Orthopaedic Foot and Ankle Society (AOFAS) hindfoot score was used to evaluate preoperative and postoperative ankle function. Results All five patients were followed up for a mean of 20 months (range: 12-24 months). The mean preoperative talo-first metatarsal angle improved from 8.46 degrees to 4.84 degrees. The preoperative mean hindfoot alignment angle was reduced from 10.9 to 5.76 degrees postoperatively. One patient had irritation due to the anchor, which needed removal after one year. Postoperatively, no patients re-experienced the feeling of "giving way". The AOFAS scores postoperatively showed two patients as excellent, two as good, and one as fair. All the patients returned to their pre-injury work. Conclusion  We have developed a technique for combined deltoid and spring ligament reconstruction using a quadrangular construct. This technique helps to restore anatomical stability, is safe, easily reproducible, and has shown positive short-term results in follow-up. The level of evidence is one of the methods used to categorize the quality and reliability of research, and our study falls under the category of level IV evidence.

Comparative transcriptome analysis, unfolding the pathways regulating the seed-size trait in cultivated lentil (Lens culinaris Medik.).

Market class, cooking time, quality, and milled grain yield are largely influenced by the seed size and shape of the lentil (Lens culinaris Medik.); thus, they are considered to be important quality traits. To unfold the pathways regulating seed size in lentils, a transcriptomic approach was performed using large-seeded (L4602) and small-seeded (L830) genotypes. The study has generated nearly 375 million high-quality reads, of which 98.70% were properly aligned to the reference genome. Among biological replicates, very high similarity in fragments per kilobase of exon per million mapped fragments values (R > 0.9) showed the consistency of RNA-seq results. Various differentially expressed genes associated mainly with the hormone signaling and cell division pathways, transcription factors, kinases, etc. were identified as having a role in cell expansion and seed growth. A total of 106,996 unigenes were used for differential expression (DE) analysis. String analysis identified various modules having certain key proteins like Ser/Thr protein kinase, seed storage protein, DNA-binding protein, microtubule-associated protein, etc. In addition, some growth and cell division-related micro-RNAs like miR3457 (cell wall formation), miR1440 (cell proliferation and cell cycles), and miR1533 (biosynthesis of plant hormones) were identified as having a role in seed size determination. Using RNA-seq data, 5254 EST-SSR primers were generated as a source for future studies aiming for the identification of linked markers. In silico validation using Genevestigator® was done for the Ser/Thr protein kinase, ethylene response factor, and Myb transcription factor genes. It is of interest that the xyloglucan endotransglucosylase gene was found differentially regulated, suggesting their role during seed development; however, at maturity, no significant differences were recorded for various cell wall parameters including cellulose, lignin, and xylose content. This is the first report on lentils that has unfolded the key seed size regulating pathways and unveiled a theoretical way for the development of lentil genotypes having customized seed sizes.

Arabidopsis GELP7 functions as a plasma membrane-localized acetyl xylan esterase, and its overexpression improves saccharification efficiency.

Acetyl substitution on the xylan chain is critical for stable interaction with cellulose and other cell wall polymers in the secondary cell wall. Xylan acetylation pattern is governed by Golgi and extracellular localized acetyl xylan esterase (AXE). We investigated the role of Arabidopsis clade Id from the GDSL esterase/lipase or GELP family in polysaccharide deacetylation. The investigation of the AtGELP7 T-DNA mutant line showed a decrease in stem esterase activity and an increase in stem acetyl content. We further generated overexpressor AtGELP7 transgenic lines, and these lines showed an increase in AXE activity and a decrease in xylan acetylation compared to wild-type plants. Therefore, we have named this enzyme as AtAXE1. The subcellular localization and immunoblot studies showed that the AtAXE1 enzyme is secreted out, associated with the plasma membrane and involved in xylan de-esterification post-synthesis. The cellulose digestibility was improved in AtAXE1 overexpressor lines without pre-treatment, after alkali and xylanases pre-treatment. Furthermore, we have also established that the AtGELP7 gene is upregulated in the overexpressor line of AtMYB46, a secondary cell wall specific transcription factor. This transcriptional regulation can drive AtGELP7 or AtAXE1 to perform de-esterification of xylan in a tissue-specific manner. Overall, these data suggest that AtGELP7 overexpression in Arabidopsis reduces xylan acetylation and improves digestibility properties of polysaccharides of stem lignocellulosic biomass.

Surgeon administered direct adductor canal block is as good as ultrasound guided adductor canal block in pain management in knee replacements- A retrospective case-control study.

Aim: The aim of this study is to assess the feasibility of the DACB in a clinical setting and compare the efficacy of postoperative pain relief after TKR in the patients administered DACB versus USG guided ACB. Also to see efficacy and safety of USACB in patients operated with medial parapatellar and subvastus approach. Material and methods: 250 consecutive patients operated with TKR between Jan 2019 to March 2022 were included. Group A included patients operated with medial parapatellar approach and received USACB, Group B included patients operated with medial parapatellar approach and received DACB while Group C included patients operated with subvastus approach and received USACB. VAS scores between three groups were compared at 12 and 24 h. All three groups of patients were otherwise treated identically in the hospital. Results: The mean age and BMI was not statistically significant between the three groups. The mean VAS pain score at rest at 12 h was 3.06 ± 1.49 (Group A) vs 1.58 ± 1.19 (Group B) [p < 0.0001] and 3.06 ± 1.49 (Group A) vs 1.88 ± 1.18 (Group C) [p < 0.0001]; and at 24 h was 1.88 ± 1.31 (Group A) vs 2.39 ± 1.27 (Group B) [p = 0.023] and 1.88 ± 1.31 (Group A) vs 2.19 ± 1.29 (Group C) [p = 0.16]. The mean theatre time was 151.9 ± 11.37 min (Group A) vs 141.02 ± 19.46 min (Group B) (p = 0.0003) and 151.9 ± 11.37 min (Group A) vs 150.4 ± 28.74 min (Group C) (p = 0.72). Hospital stay was 3.82 ± 0.80 (Group A) vs 4.0 ± 1.09 (Group B) [p = 0.30] and 3.82 ± 0.80 (Group A) vs 2.7 ± 0.69 (Group C) [p < 0.0001]. Group B and Group C had one complication each. Conclusion: USG ACB irrespective of approach used remains the gold standard in providing consistent pain relief and thereby facilitating early discharge. However, increased operating room turnover time and repeated top-ups remain a disadvantage. Both the quantum of pain relief and the potential downsides remained the same irrespective of the surgical approach used and whether or not steroid was added to the cocktail used for infiltration. On the other hand, DACB provides a short lasting (24 h) adequate pain relief after TKR with similar low complication rates. The technique of DACB may have a potential for a wider use especially in centres where outpatient arthroplasties are performed, if newer longer acting anaesthetic/analgesic combinations are devised.

Comparative Study of Pinless Navigation System versus Conventional Instrumentation in Total Knee Arthroplasty.

BACKGROUD: Optimal placement of the components and achieving a neutral mechanical axis are the main goals of total knee arthroplasty (TKA). Different computerised navigation systems are presently used for these purposes. This aim of this study was to compare the pinless navigation (PNA) TKA performed using iAssist with the conventional instrumented (CIN) TKA in terms of functional and radiological outcomes. METHODS: A total of 100 knees operated for TKA by a single surgeon were studied retrospectively for a period of 2 years. Weight-bearing postoperative radiographs of the knees along with scanograms of the lower limbs were used for measurements of component positioning, mechanical axis alignment, and number of outliers. Oxford knee scoring was used for functional analysis. RESULTS: No statistically significant difference was seen in the mean mechanical axis alignment (hip-knee-ankle angle), coronal alignment (α and ß angles) and sagittal alignment (γ and δ angles) of the femoral and tibial components between the two groups. Though the percentage of outliers for mechanical axis alignment was lower in the PNA-TKA group than in the CIN-TKA group, the difference was not statistically significant (p = 0.73). The number of outliers for the femoral and tibial component positioning in coronal and sagittal planes was not statistically significantly different between the two groups. No statistically significant difference (p = 0.68) was noted between the two groups with respect to the Oxford Knee Score. The mean surgical time was greater in the PNA-TKA group by 11 minutes, which was statistically significantly longer (p = 0.018). Complications were seen in 6.89% of the cases in the CIN-TKA group, while none in the PNA-TKA group. CONCLUSIONS: The accurate mechanical axis alignment and component positioning can be achieved with the conventional instrumentation, so the use of PNA system, which adds to the surgical cost, is questionable. Also, equally good short-term functional outcome can be achieved with the conventional instrumentation. The surgeon must be accustomed with the instrumentation of the PNA system, or it adds to the surgical time.

Is left atrial fibrosis an independent determinant of atrial fibrillation in mitral stenosis?

We prospectively studied whether left atrial (LA) fibrosis is a determinant of atrial fibrillation (AF) in mitral stenosis in patients who underwent balloon mitral valvotomy. There were 2 groups: Group A (n = 16), with AF and Group B (n = 27), without AF. Fibrosis was assessed by MRI. Patients underwent cardioversion before MRI. There were 27 females and 16 males, aged 29 ± 6 years. The LA areas in Groups A and B were 54.3 ± 4.4 mm2 and 39.4 ± 2.3 mm2 (p < 0.05) and the LA volume index was 46.2 ± 2.9 ml/m2 vs 33 ± 3 ml/m2 respectively (p < 0.0001). The presence of LA scarring was not statistically different in the two groups.

Roof arc width: The novel calculation method for calculation of patient specific roof arc width in acetabular fractures.

PURPOSE: Roof arc angle (RAA) is determined by measuring angle between a vertical line drawn from center of the acetabulum towards the acetabular dome and a second line drawn from center of acetabulum to the fracture through the acetabulum. Joel and Matta demonstrated that when roof arc angle was less than 45° on Pelvic AP and Judet's views, the fracture line is considered to be passing through the weight-bearing dome and require surgical fixation. The main purpose of the study is to calculate patient-specific angle and width for the better evaluation and management of acetabular fractures. METHODS: Radiographs of normal hips were retrieved from electronic data and parameters were calculated. Two observers calculated the parameters at two different intervals. Pearson correlation formula was used to find a correlation between groups. RESULTS: Fifty radiographs of 28 patients were reviewed. The mean age of patients was 75.58 years ±13.28. The radius of the acetabulum, the radiographic measurement of sector width for 45° angulation at the roof, and the mathematical calculation for roof arc for 45° angle had significant positive correlation for both observers at two different occasions. CONCLUSIONS: The measured roof arc width ranges from a minimum of 16.20 mm-31.50 mm and the calculated arc width for a 45-degree angle varies from a minimum of 15 mm-25.56 mm. These values are higher than the described values of 10 mm equals to 45 degrees. Hence, the values measured in this study should be considered for decision making in the management of acetabular fractures.

Robotic-Assisted Knee Arthroplasty (RAKA): The Technique, the Technology and the Transition.

BACKGROUND: There has been an exponential increase in knee arthroplasty over the past 20 years. This has led to a quest for improvement in outcomes and patient satisfaction. While the last decade of last century proved to be the decade for Computer-Assisted Surgery (CAS) or Computer Navigation wherein the technology demonstrated a clear benefit in terms of improving mechanical axis alignment and component positioning, this decade is likely to belong to Robotics. Robotics adds an independent dimension to the benefits that CAS offers. The article deals with the generation of robots, technical steps in robotics, advantages and downsides of robotics and way forward in the field of knee arthroplasty. MATERIALS AND METHODS: The review article was designed and edited by six different authors reviewing 32 relevant pubmed-based articles related to robotics in arthroplasty and orthopaedics. The concept, design and the definition of the intellectual content were based on the internationally published literature and insightful articles. The review is also based on the clinical experimental studies published in the literature. DISCUSSION: The robotic arm is actively involved with surgeon to achieve the precision and outcomes that the surgeon aims for. With the concept of haptic boundaries and augmented reality being incorporated in most systems, Robotic Assisted Arthroplasty (RAA) is likely to offer several advantages. The potential advantages of these systems may include accuracy in gap balancing, component positioning, minimal bone resection, reduced soft tissue handling and trauma, patient anatomy specific resection, and real time feedback. They, however, come with their own downsides in terms of capital cost, learning curve, time consumption and unclear advantages in term of long-term clinical outcomes. CONCLUSION: To conclude, this review article offers a balanced view on how the technology is impacting current arthroplasty practice and what can be expected in coming years. The commitment of almost all major implant manufacturers in investing in robotics likely means that the evolution of Robotic technology and this decade will be exciting with rapid strides revealing paradigm shift and evolution of technology with significant reductions of cost enabling it to be available universally. For technology to populate in operating room, I think it will be result of exposure of young surgeons to these computers and robotics, as they grow in with confidence with technology from residency days to offer better precision in future.

Retro-auricular endo assisted selective neck dissection (how I do it).

Elective neck dissection (END) is preferred in all treatment naïve patients with invasive oral squamous cell carcinoma (OSCC), including the early stage, node negative cases (T1/T2 N0). Usually the conventional horizontal neck crease incision leads to a faintly visible scar in the neck. However sometimes, the neck scar is hypertrophic and is highly unaesthetic and psychologically distressing to the patient. Retro-auricular hairline approach has been popularized in the Robot assisted neck dissections (RoAND), to avoid easily visible scar in neck crease. We have been using the retro-auricular incision for selective neck dissection in early invasive OSCCs using an assembly of customized retractors as an open approach with only occasional use of endoscopes. This approach to the neck surgery is oncologically safe, reproducible and economical. It is cosmetically superior as the scar of neck dissection is in a less conspicuous area of the face, hidden behind the ear and in the hairline.

Cell Wall Acetylation in Hybrid Aspen Affects Field Performance, Foliar Phenolic Composition and Resistance to Biological Stress Factors in a Construct-Dependent Fashion.

The production of biofuels and "green" chemicals from the lignocellulose of fast-growing hardwood species is hampered by extensive acetylation of xylan. Different strategies have been implemented to reduce xylan acetylation, resulting in transgenic plants that show good growth in the greenhouse, improved saccharification and fermentation, but the field performance of such plants has not yet been reported. The aim of this study was to evaluate the impact of reduced acetylation on field productivity and identify the best strategies for decreasing acetylation. Growth and biological stress data were evaluated for 18 hybrid aspen lines with 10-20% reductions in the cell wall acetyl content from a five year field experiment in Southern Sweden. The reduction in acetyl content was achieved either by suppressing the process of acetylation in the Golgi by reducing expression of REDUCED WALL ACETYLATION (RWA) genes, or by post-synthetic acetyl removal by fungal acetyl xylan esterases (AXEs) from two different families, CE1 and CE5, targeting them to cell walls. Transgene expression was regulated by either a constitutive promoter (35S) or a wood-specific promoter (WP). For the majority of transgenic lines, growth was either similar to that in WT and transgenic control (WP:GUS) plants, or slightly reduced. The slight reduction was observed in the AXE-expressing lines regulated by the 35S promoter, not those with the WP promoter which limits expression to cells developing secondary walls. Expressing AXEs regulated by the 35S promoter resulted in increased foliar arthropod chewing, and altered condensed tannins and salicinoid phenolic glucosides (SPGs) profiles. Greater growth inhibition was observed in the case of CE5 than with CE1 AXE, and it was associated with increased foliar necrosis and distinct SPG profiles, suggesting that CE5 AXE could be recognized by the pathogen-associated molecular pattern system. For each of three different constructs, there was a line with dwarfism and growth abnormalities, suggesting random genetic/epigenetic changes. This high frequency of dwarfism (17%) is suggestive of a link between acetyl metabolism and chromatin function. These data represent the first evaluation of acetyl-reduced plants from the field, indicating some possible pitfalls, and identifying the best strategies, when developing highly productive acetyl-reduced feedstocks.

First record of off-season flowering in Populus deltoides from India: paradigm of climate change indicator.

Populus deltoides is a fast-growing woody species possessing plethora of industrial applications. This species evolutionarily developed unisexual male and female catkin inflorescence on separate trees. Flowering usually occurs during early spring before the development of foliage, where buds appear near axils or at the extending shoots. In 2019, surveys were undertaken to study the flowering pattern of P. deltoides in the states of Punjab, Haryana, Uttar Pradesh and Uttarakhand in northern India. Interestingly, an anomalous flowering behaviour (appearance of off-season male catkins during autumn, i.e. October) was observed in a plantation trial at Kapurthala, Punjab. The male catkins were 2.7-3.1 ± 0.07 cm long and 0.3-0.5 ± 0.03 cm wide, which is significant for flowering and liberation of pollen grains. Preliminary results suggested that climatic factors, such as episodes of high or low temperature and the precipitation variation forcing the tree species to behave differently. Unearthing the climate-driven off-season flowering in other tree species alluded the stimulation of phytohormones, such as gibberellic and salicylic acid concentrations influencing the flowering time, therefore, needs further investigation in case of P. deltoides. Overall, this work provides early clues of changing climatic scenario altering the flowering pattern of a tropical forestry tree species.

Hybrid Aspen Expressing a Carbohydrate Esterase Family 5 Acetyl Xylan Esterase Under Control of a Wood-Specific Promoter Shows Improved Saccharification.

Fast-growing broad-leaf tree species can serve as feedstocks for production of bio-based chemicals and fuels through biochemical conversion of wood to monosaccharides. This conversion is hampered by the xylan acetylation pattern. To reduce xylan acetylation in the wood, the Hypocrea jecorina acetyl xylan esterase (HjAXE) from carbohydrate esterase (CE) family 5 was expressed in hybrid aspen under the control of the wood-specific PtGT43B promoter and targeted to the secretory pathway. The enzyme was predicted to deacetylate polymeric xylan in the vicinity of cellulose due to the presence of a cellulose-binding module. Cell-wall-bound protein fractions from developing wood of transgenic plants were capable of releasing acetyl from finely ground wood powder, indicative of active AXE present in cell walls of these plants, whereas no such activity was detected in wild-type plants. The transgenic lines grew in height and diameter as well as wild-type trees, whereas their internodes were slightly shorter, indicating higher leaf production. The average acetyl content in the wood of these lines was reduced by 13%, mainly due to reductions in di-acetylated xylose units, and in C-2 and C-3 mono-acetylated xylose units. Analysis of soluble cell wall polysaccharides revealed a 4% reduction in the fraction of xylose units and an 18% increase in the fraction of glucose units, whereas the contents of cellulose and lignin were not affected. Enzymatic saccharification of wood from transgenic plants resulted in 27% higher glucose yield than for wild-type plants. Brunauer-Emmett-Teller (BET) analysis and Simons' staining pointed toward larger surface area and improved cellulose accessibility for wood from transgenic plants compared to wood from wild-type plants, which could be achieved by HjAXE deacetylating xylan bound to cellulose. The results show that CE5 family can serve as a source of enzymes for in planta reduction of recalcitrance to saccharification.

Mitochondrial cytochrome oxidase C subunit III (cox3) gene as a sensitive and specific target for molecular detection of Babesia gibsoni infection in dogs.

A PCR targeting mitochondrial cytochrome oxidase subunit III (cox3) for molecular detection of Babesia gibsoni infection in dogs has been developed in this study. Fifty blood samples from suspected clinical cases from dogs, brought to the veterinary college clinics, were examined for presence of B. gibsoni using conventional diagnosis by microscopic examination of Giemsa stained thin blood smears. In addition, species specific PCRs targeting ITS-1 region (BgITS-1 PCR) and nested PCR targeting 18S ribosomal RNA gene (Bg18SnPCR) were carried out. A 634 bp PCR fragment of B. gibsoni cox3 gene was amplified in positive samples from three geographical locations of Satara, Wai and Pune in Maharashtra state of India. From analysis of the sequence of the B. gibsoni cox3 gene, we found that the Indian isolate had 96-98% similarity to the isolate from Japan and China. Post sequencing, de-novo diagnostic primer pair for species specific amplification of 164 bp fragment of B. gibsonicox3 was designed and the PCR was standardized. The diagnostic results of de-novo Bgcox3 PCR were compared with BgITS-1 PCR and Bg18S nPCR. Thin blood smears detected 22% (11/50) samples positive for small form of Babesia species. The BgITS-1 PCR detected 25% samples (15/50) as positive and Bg18S nPCR detected 80% (40/50) B. gibsoni positive samples. The de-novo Bgcox3 PCR detected 66% (33/50) samples positive for B. gibsoni (at 95% CI). The analytical sensitivity of cox3 PCR was evaluated as 0.000003% parasitaemia or 09 parasites in 100  µl of blood. The de-novo diagnostic cox3 PCR did not cross react with control positive DNA from other haemoprotozoa and rickettsia like B. vogeli, Hepatozoon canis, Trypanosoma evansi, Ehrlichia canis and Anaplasma platys. Statistically, cox3 PCR had better diagnostic efficiency than ITS-1 PCR in terms of sensitivity (p = 0.0006). No statistically significant difference between results of cox3 PCR and 18S nPCR was observed (p = 0.1760). Kappa values estimated for each test pair showed fair to moderate agreement between the observations. Specificity of Bgcox3 PCR was 100% when compared with microscopy or BgITS-1 PCR. Sensitivity of Bgcox3 PCR was 100% when compared with that of Bg18S nPCR.

Evaluation and Correlation of Multiple Anthelmintic Resistances to Gastrointestinal Nematodes Using Different Fecal Egg Count Reduction Methods in Small Ruminants of Punjab, India.

PURPOSE: Concordance of multiple anthelmintic resistances for gastrointestinal nematodes in small ruminants by three average-based and two individually based fecal egg count reduction (FECR) tests was evaluated and corrected. METHODS: Sheep and goats (≥ 8 weeks) from five farms were randomly assigned to three treatment groups (I, II, III; n = 10 per group) and one untreated control group (Group IV; n = 10). Group I received fenbendazole at the dose rate of 5 and 10 mg/kg, Group II received ivermectin at the dose rate of 0.2 and 0.4 mg/kg, and Group III received levamisole at the dose rate of 8 and 12 mg/kg body weight orally for sheep and goat, respectively. Three average-based methods of FECR (FECR1, FECR2 and FECR3) and two individually based methods of FECR (iFECR1 and iFECR2) were evaluated. RESULTS: For fenbendazole resistance, Spearman correlation coefficient for FECR1 was non-significant with other formulae, but for FECR2 with FECR3, FECR3 with iFECR1 and iFECR1 with iFECR2 coincidence was significant at 1%, while for FECR2 with iFECR2 and FECR3 with iFECR2 it was significant at 5%. Spearman correlation coefficients for ivermectin resistance were significant at 1% level and for levamisole it showed significant coincidence at 1% for FECR1 with FECR2 and iFECR1, FECR2 with FECR3 and iFECR1, and iFECR1 with iFECR2, while for FECR1 with FECR3 and iFECR2 coincidence was significant at 5% level. Concordance of kappa values indicated that the coincidence of the prevalence of anthelmintic resistance (95% CI) among the five farms was non-significant. CONCLUSIONS: Concordance between the standard average-based FECR and individually based methods suggests that either method could be applied to small ruminant farms.

A proposed radiological classification system of Hoffa's fracture based on fracture configuration and consequent optimal treatment strategy along with the review of literature.

BACKGROUND: Coronal fractures of distal end femur, referred as Hoffa's fracture are not uncommon, yet easily missed injuries lacking proper classification system and consensus for ideal treatment. While most trauma surgeons adopt different strategies based on the fracture configuration and their own experience, there are no set ways to classify these based on the most appropriate treatment strategy. METHODS: Thirty cases of Hoffa fracture from tertiary care centres were studied for the fracture pattern, fragment size, comminution and possible variations to formulate a radiological classification and treatment guidelines. Additionally, a literature search was used to analyze 77 case studies based on Hoffa fracture to find out the common fracture patterns and treatment modalities adopted for varying fracture patterns in these studies. Six independent observers participated in testing the inter-observer reliability of the proposed classification. RESULTS: A new proposed radiological classification for Hoffa fracture consists of four main types. Type 1 is with fracture fragment >2.5 cm, Type 2 with fragment <2.5 cm, Type 3 is comminuted fracture, Type 4 are subdivided as Type 4a - Anterior, Type 4b - Bicondylar, Type 4c - Osteochondral type and Type 4d - With supracondylar extension. Optimum treatment modality depends on the type of Hoffa's fracture and has been suggested in the study. Interobserver reliability demonstrated that overall agreement was 0.907692 with a fixed marginal Kappa of 0.881067 and free Marginal Kappa at 0.892308. Intra-observer reliability test for the classification system showed a strong Kappa value of +1.0. CONCLUSION: The new suggested classification helps identify different types of Hoffa's fracture. This is likely to help decide optimal surgical treatment depending on the nature of the injury. The classification system has high inter and intra-observer reliability that enables its universal applicability.

Intermediate term outcome after electrogram guided segmental ostial pulmonary vein isolation using an 8 mm tip catheter for paroxysmal atrial fibrillation.

INTRODUCTION: Pulmonary vein isolation (PVI) is the most widely used procedure for ablation in patients with paroxysmal atrial fibrillation (AF). Not withstanding recent advancements in this field, including sophisticated three-dimensional (3D) based imaging and advanced ablation catheters with contact force technology, many patients and healthcare systems in developing countries will not afford such an expensive therapeutic procedure. There are no data from India analyzing the efficacy of PVI for PAF using conventional mapping and ablation. In this article, we have summarized the intermediate term outcome following PVI in patients with PAF using electrogram-based mapping and a 8 mm tip ablation catheter. METHOD: A total of 42 consecutive patients who underwent PVI for symptomatic PAF not controlled with at least one antiarrhythmic drug were studied in a tertiary care institute from March 2011 to June 2018. Patients with rheumatic AF were excluded. The pulmonary vein (PV) anatomy was assessed by pulmonary angiography during the ablation procedure. Using conventional electrophysiologic mapping, a variable curve Lasso catheter placed in the PVs was used to guide the earliest site of breakthrough. The segmental ostial PVI was performed using a 8 mm tip radiofrequency (RF) ablation catheter. Elimination of all PV ostial potentials and complete entrance block into the PV were considered indicative of complete electrical isolation. Follow-up visits were scheduled at one, three, and six months after the procedure, and every six months thereafter. History, symptom review, clinical examination, and 12-lead ECG were performed at each follow-up. RESULTS: At pre-discharge, 34 patients (81%) were in sinus rhythm, while eight patients (19%) continued to have atrial fibrillation. The age of the study population was 51.5 ± 11.7 yrs. The mean follow-up duration was 44 ± 21 months (range 6-84 months). The number of PVs isolated included one (five patients, 11.9%), two (20 patients, 47.6%), three (12 patients, 28.6%), and four (five patients, 11.9%). In 42 patients, a total of 101 PVs were isolated. The right superior PV (RSPV) was isolated in 37 patients, the left superior PV (LSPV) was isolated in 39 patients, the left inferior PV (LIPV) was isolated in 14 patients, and the right inferior PV (RIPV) was isolated in six patients. The procedure duration was 125 ± 29 min and the fluoroscopy time was 47 ± 13 min. The number of patients who remained in sinus rhythm at 1, 6, 12, and 24 months were 34 (81%), 32 (76%), 30 (71%), and 26 (62%), respectively. Two patients of these underwent repeat PVI, which was successful, and they had freedom from AF episodes. Complications were rare. One patient had a minor pericardial effusion, and one patient had transient sinus pauses, which were conservatively managed. CONCLUSION: Conventional RF ablation using PV potential-based mapping and ablation with 8 mm tip catheters is safe for patients with PAF. The intermediate term outcome is satisfactory and cost-effective in our setting with limited resources.