The cascade screening in heritable forms of pulmonary arterial hypertension.

Heritable pulmonary artery hypertension (HPAH) is an increasingly recognized type of pulmonary arterial hypertension, in both pediatric and adult population. Intrinsic to hereditary disease, screening for genetic mutations within families is an important component of diagnosis and understanding burden of disease. Recently, consensus guidelines are published for genetic screening in PAH. These guidelines include recommendations for screening at diagnosis, noting individuals with presumed PAH due to familial, or idiopathic etiologies. Cascade genetic testing is specifically recommended as a testing paradigm to screen relatives for detection of mutation carriers, who may be asymptomatic. Without targeted genetic testing, familial mutation carriers may only come to attention when pulmonary vascular disease burden is high enough to cause symptoms, suggesting more advanced disease. Here, we present our collective experience with HPAH in five distinct families, specifically to report on the clinical courses of patients who were diagnosed with genetic mutation at diagnosis versus those who were offered genetic screening. In three families, asymptomatic mutation carriers were identified and monitored for clinical worsening. In two families, screening was not done and affected family members presented with advanced disease.

Interlink between ExoD (Alr2882), exopolysaccharide synthesis and metal tolerance in Nostoc sp. strain PCC 7120: Insight into its role, paralogs and evolution.

Exopolysaccharides (EPS) produced by bacterial species are an important component of bacteria's survival strategy. Synthesis of EPS, principal component of extracellular polymeric substance, occurs through multiple pathways involving multitude of genes. While stress-induced concomitant increase in exoD transcript levels and EPS content have been shown earlier, experimental evidence for direct correlation is lacking. In the present study, role of ExoD in Nostoc sp. strain PCC 7120 was evaluated by generating a recombinant Nostoc strain AnexoD+, wherein the ExoD (Alr2882) protein was constitutively overexpressed. AnexoD+ exhibited higher EPS production, propensity for formation of biofilms and tolerance to Cd stress compared to vector control AnpAM cells. Both Alr2882 and its paralog All1787 exhibited 5 transmembrane domains, with only All1787 predicted to interact with several proteins in polysaccharide synthesis. Phylogenetic analysis of orthologs of these proteins across cyanobacteria indicated that the two paralogs Alr2882 and All1787 and their corresponding orthologs arose divergently during evolution, and could have distinct roles to perform in the biosynthesis of EPS. This study has thrown open the possibility of engineering overproduction of EPS and inducing biofilm formation through genetic manipulation of EPS biosynthesis genes in cyanobacteria, thus building a cost-effective green platform for large scale production of EPS.

Cyclic peptides nanospheres: A '2-in-1' self-assembled delivery system for targeting nucleus and cytoplasm.

Vascular endothelial growth factor (VEGF) is considered as one of the vital growth factors for angiogenesis, which is primarily responsible for the progress and maintenance of new vascular network in tumor. Numerous studies report that inhibition of VEGF-induced angiogenesis is a potent technique for cancer suppression. Recently, RNA interference, especially small interfering RNA (siRNA) signified a promising approach to suppress the gene expression. However, the clinical implementation of biological macromolecules such as siRNA is significantly limited because of stability and bioavailability issues. Herein, self-assembled peptide nanospheres have been generated from L,L-cyclic peptides using hydrophobic (Trp), positively charged (Arg) and cysteine (Cys) amino acid residues and demonstrated as vehicles for intracellular delivery of VEGF siRNA and VEGF antisense oligonucleotide. Formation of peptide nanostructures is confirmed by HR-TEM, AFM, SEM and DLS analysis. Possible mechanism of self-assembly of the cyclic peptides and their binding with macromolecules are demonstrated by in-silico analysis. Gel electrophoresis reveals that the newly generated peptide based organic materials exhibit strong binding affinity toward siRNAs / antisense oligonucleotides (ASOs) at optimum concentration. Flow cytometry and confocal microscopy results confirm the efficiency of the new biomaterials toward the intracellular delivery of fluorescent labeled siRNA / ASOs. Furthermore, VEGF expression evaluated by western blot and RT-PCR upon the delivery of functional VEGF siRNA/ASOs suggests that very low concentrations of VEGF siRNA/ASOs cause significant gene knockdown at protein and mRNA levels, respectively.

Cloning, expression and characterization of P1 and NIa proteases from banana bract mosaic virus (BBrMV).

Banana bract mosaic virus (BBrMV) causes the banana bract mosaic disease in banana. It belongs to the genus Potyvirus within the family Potyviridae. To the best of our knowledge apart from BBrMV coat protein gene, there are no reports on cloning, expression and characterization of any other genes from BBrMV. In this study, the BBrMV P1 and NIa protease genes were amplified from BBrMV infected banana plant cultivar Nendran and were cloned into the protein expression vector pET28b. Recombinant plasmids were transferred to BL21-CodonPlus (DE3)-RP cells and the IPTG (Isopropyl ß-d-1-thiogalactopyranoside) induced BBrMV P1 and NIa proteins with molecular weights of 42 and 32 KDa respectively were purified on Ni-NTA resin column under denaturing conditions using 8 M urea. BBrMV P1 and NIa purified proteins were detected by Western blot using anti-histidine antibody. The activity of both P1 and NIa proteases in native form was analyzed through in-gel zymographic assay. The activities of both the proteases were strongly inhibited by PMSF, suggesting that both the proteases are the serine type proteases. Interestingly both the proteases showed a temperature optimum of 50 °C while the pH optimum was 8. Both proteases lost their activity when incubated at 70 °C for 1 h. This is the first report of expression, purification and characterization of BBrMV P1 and NIa proteases.

Analysis of synonymous codon usage bias and phylogeny of coat protein gene in banana bract mosaic virus isolates.

The banana is one of the world's most important livelihood crops. Banana plants are principally infected by four virus species, Banana bunchy top virus (genus Babuvirus), Cucumber mosaic virus (genus Cucumovirus), Banana streak virus (genus Badnavirus) and Banana bract mosaic virus (genus Potyvirus). The objective of this study is to understand the codon usage pattern and phylogeny of coat protein gene in different banana bract mosaic virus (BBrMV) isolates. The BBrMV Coat Protein (CP) gene was amplified from BBrMV infected banana plant samples collected from different districts of Tamil Nadu and Karnataka, India. Six new BBrMV isolates were submitted to National Center for Biotechnology Information. Phylogenetic analysis and codon usage indices were studied along with other isolates of BBrMV. Phylogenetic analysis of CP genes shows that most of BBrMV isolates are closely related to each other except KF385484.1 and KF385478.1. Relative codon usage patterns among different BBrMV isolates were calculated by software CodonW version 1.4.2. In BBrMV, codons with A-ended or U ended are the most preferential except the Leu and Gln whose optimized codons are CAG and UUG ending by G. The codon usage patterns of BBrMV isolates are principally influenced by mutational bias; however, compositional constraints along with mutational bias also play a major role.

Knowledge and attitude about sexually transmitted infections other than HIV among college students.

BACKGROUND: Sexually transmitted infections (STIs) are the infections which are mainly transmitted through sexual intercourse. Young individuals in the age group of 16 to 24 years are considered to be at more risk for STIs compared to older adults. Young individuals are more likely to practice unprotected sex and have multiple sexual partners. If the STIs are not treated adequately, it can lead to various complications.Most of the people may be aware about HIV/AIDs because of the awareness created by media and the government programs, however knowledge about STIs other than HIV/AIDS is low in the developing countries. MATERIALS AND METHODS: This study was a descriptive cross sectional study to assess the knowledge, awareness and attitude of college students about STIs other than HIV. A total of 350 engineering students from various semesters were included in the study. They were asked to fill up an anonymous questionnaire. RESULTS: Two hundred and fifty six (73%) males and 94 (27%) females participated in the study. 313 (90%) students had heard about sexually transmitted infections (STIs) and 223 (64%) students had heard about STIs other than HIV. 99% of students knew about HIV where as less than 50% of students knew about other STIs. Teachers, internet and media were the source of information for most of the participants. Almost 75% of the students knew about the modes of transmission of STIs. Less than 50% of the participants knew about the symptoms of STIs and complications. Also attitude of the students towards sexual health and prevention of STIs was variable. CONCLUSION: The findings of our study shows that it is important to orient the students about sexual health and safe sexual practices as it will go a long way in prevention and control of STIs. Also the morbidities and complications associated with STIs can be prevented.

Comparative analysis of MazEF and HicAB toxin-antitoxin systems of the cyanobacterium, Anabaena sp. PCC7120.

Anabaena PCC7120 has two annotated toxin-antitoxin systems: MazEF and HicAB. Overexpression of either of the toxins severely inhibited the growth of Escherichia coli BL21(plysS)(DE3). Of the two Anabaena toxins, MazF exhibited higher toxicity than HicA as evidenced by (i) 100-fold lower viability upon overexpression of MazF compared to HicA; (ii) complete loss of cell viability within 1 h of induction of MazF expression, as against >103 colony forming units mL-1 in case of HicA; (iii) inability to maintain the MazF overexpressing plasmid in E. coli cells; and (iv) neutralisation of the toxin was effective at the molar ratio of 1:1.9 for MazF:MazE and 13:1 for HicA:HicB, indicating higher antitoxin requirement for neutralisation of MazF. The growth inhibitory effect of MazF was found to be higher in lag phase cultures compared to mid-logarithmic phase cultures of E. coli, while the reverse was true for HicA. The results suggest possible distinct roles for MazEF and HicAB systems of Anabaena.

Topical Steroid Awareness and Abuse: A Prospective Study among Dermatology Outpatients.

BACKGROUND: Topical steroids are one of the most commonly abused drugs. There are only a few studies available which have highlighted the severity of this problem in India. However, these studies have concentrated mainly on the topical steroid abuse and its side effects over the face. AIMS: The aim of this study was to know the awareness among the people about various commonly available topical steroids and their combinations irrespective of usage and to know the extent of misuse of these drugs. Along with this, we also tried to find the source of recommendation of these medicines which will help to sensitize people about this menace. MATERIALS AND METHODS: This was a prospective, questionnaire-based study done at a tertiary care hospital. A total of 1000 adult patients attending the dermatology outpatient department were administered a questionnaire about awareness and usage of topical steroid and its combinations. RESULTS: A total of 1000 adult patients were included in the study, out of which 809 (80.9%) patients had heard about at least one of the topical steroids or its combinations mentioned in the questionnaire. Six hundred and twelve (61.2%) patients had used these creams. Acne and pigmentation were the most common indications for which topical steroid was used. These medicines were recommended by general practitioners in 302 (49.5%) patients and pharmacists in 71 (11.6%) patients. Totally, 318 (51.9%) patients complained of some form of side effect after using these creams. Aggravation of the symptoms and increased pigmentation were the most common adverse effects. CONCLUSION: Misuse of topical steroids not just over the face but also as a cream for any skin problem is quite common. Most of the times, it is recommended by general practitioners or pharmacists. It is very important to sensitize these people about the possible complications of these drugs and the extent of problem the society is facing because of irrational and unregulated use of these drugs.

Calcifying Epithelial Odontogenic Tumour of the Mandible: An Unusually Aggressive Presentation of an Indolent Tumour.

Calcifying Epithelial Odontogenic Tumour (CEOT) or Pindborg tumour is a rare odontogenic tumour of epithelial origin. They constitute less than 1% of odontogenic tumours. Intra-ossseous variant of CEOT are more common compared to extra-osseous variant. Although benign, these can exhibit deceptively aggressive presentation. Here we report a rare case of CEOT in a 36-year-old female patient who presented with aggressive intra-osseous lesion with cortical breach and exuberant soft tissue proliferation. The lesion was treated with resection and reconstructed with titanium reconstruction plate.

Acoustic Transmission Characteristics of a Eustachian Tube Volitionally Opened in Two Living Subjects.

OBJECTIVE: To assess the acoustic transmission characteristics of the Eustachian tube (ET) in living subjects in verified patent and closed ET states to facilitate the detection and quantification of ET function using acoustic measures such as sonotubometry. PATIENTS: The two subjects in this study had no history of ear disease nor previous ear surgery and were capable of volitionally opening and closing their ET. INTERVENTIONS: Tympanometry and otologic examinations were used to confirm ET patent and closed states by observing tympanic membrane movement with respiration and by acoustic immitance measurements during forced respiration. A series of 500-ms long chirps containing frequencies from 100 Hz to 10 kHz were introduced into the nasal cavity during both ET states and recorded by microphones in both the contralateral naris and external auditory canal. MAIN OUTCOME MEASURES: Acoustic energy transmission through the ET across the 0.1 to 10 kHz frequency range in the closed state versus the patent state. RESULTS: An increase in acoustic energy transmission occurs across the frequencies of 1 to 4 kHz between the closed and patent ET states, particularly in frequencies below 2.5 kHz. CONCLUSIONS: Results support sonotubometry as a potential diagnostic tool for ET dysfunction. Acoustic differences between the ET states manifest as a general increase in transmitted signal amplitude. Characterizing the acoustic properties in the verified patent and closed ET states allows investigators to more reliably interpret sonotubometric tests of ET function.

GroEL of the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31 exhibits GroES and ATP-independent refolding activity.

The nitrogen-fixing cyanobacterium, Anabaena L-31 has two Hsp60 proteins, 59 kDa GroEL coded by the second gene of groESL operon and 61 kDa Cpn60 coded by cpn60 gene. Anabaena GroEL formed stable higher oligomer (>12-mer) in the presence of K(+) and prevented thermal aggregation of malate dehydrogenase (MDH). Using three protein substrates (MDH, All1541 and green fluorescent protein), it was found that the refolding activity of Anabaena GroEL was lower than that of Escherichia coli GroEL, but independent of both GroES and ATP. This correlated with in vivo data. GroEL exhibited ATPase activity which was enhanced in the presence of GroES and absence of a denatured protein, contrary to that observed for bacterial GroEL. However, a significant role for ATP could not be ascertained during in vitro folding assays. The monomeric Cpn60 exhibited much lower refolding activity than GroEL, unaffected by GroES and ATP. In vitro studies revealed inhibition of the refolding activity of Anabaena GroEL by Cpn60, which could be due to their different oligomeric status. The role of GroES and ATP may have been added during the course of evolution from the ancient cyanobacteria to modern day bacteria enhancing the refolding ability and ensuring wider scope of substrates for GroEL.

Adaptation of tibial structure and strength to axial compression depends on loading history in both C57BL/6 and BALB/c mice.

Tibial compression can increase murine bone mass. However, loading protocols and mouse strains differ between studies, which may contribute to conflicting results. We hypothesized that bone accrual is influenced more by loading history than by mouse strain or animal handling. The right tibiae of 4-month-old C57BL/6 and BALB/c mice were subjected to axial compression (10 N, 3 days/week, 6 weeks). Left tibiae served as contralateral controls to calculate relative changes: (loaded - control)/control. The WashU protocol applied 60 cycles/day, at 2 Hz, with a 10-s rest-insertion between cycles; the Cornell/HSS protocol applied 1,200 cycles/day, at 6.7 Hz, with a 0.1-s rest-insertion. Because sham loading, sedation, and transportation did not affect tibial morphology, unhandled mice served as age-matched controls (AC). Both loading protocols were anabolic for cortical bone, but Cornell/HSS loading elicited a more rapid response that was greater than WashU loading by 13 %. By 6 weeks, cortical bone volume of each loading group was greater than of AC (average + 16 %) and not different from each other. Ultimate displacement and energy to fracture were greater in tibiae loaded by either protocol, and ultimate force was greater with Cornell/HSS loading. At 6 weeks, independent of mouse strain, the WashU protocol produced minimal trabecular bone and the trabecular bone volume fraction of Cornell/HSS tibiae was greater than that of AC by 65 % and that of WashU by 44 %. We concluded that tibial adaptation to loading was more influenced by waveform than mouse strain or animal handling and therefore may have targeted similar osteogenic mechanisms in C57BL/6 and BALB/c mice.

Short-term low-strain vibration enhances chemo-transport yet does not stimulate osteogenic gene expression or cortical bone formation in adult mice.

Development of low-magnitude mechanical stimulation (LMMS) based treatment strategies for a variety of orthopaedic issues requires better understanding of mechano-transduction and bone adaptation. Our overall goal was to study the tissue and molecular level changes in cortical bone in response to low-strain vibration (LSV: 70 Hz, 0.5 g, 300 µÎµ) and compare these to changes in response to a known anabolic stimulus: high-strain compression (HSC: rest inserted loading, 1000 µÎµ). Adult (6-7 months) C57BL/6 mice were used for the study and non-invasive axial compression of the tibia was used as a loading model. We first studied bone adaptation at the tibial mid-diaphysis, using dynamic histomorphometry, in response to daily loading of 15 min LSV or 60 cycles HSC for 5 consecutive days. We found that bone formation rate and mineral apposition rate were significantly increased in response to HSC but not LSV. The second aim was to compare chemo-transport in response to 5 min of LSV versus 5 min (30 cycles) of HSC. Chemo-transport increased significantly in response to both loading stimuli, particularly in the medial and the lateral quadrants of the cross section. Finally, we evaluated the expression of genes related to mechano-responsiveness, osteoblast differentiation, and matrix mineralization in tibias subjected to 15 min LSV or 60 cycles HSC for 1 day (4-h time point) or 4 consecutive days (4-day time point). The expression level of most of the genes remained unchanged in response to LSV at both time points. In contrast, the expression level of all the genes changed significantly in response to HSC at the 4-h time point. We conclude that short-term, low-strain vibration results in increased chemo-transport, yet does not stimulate an increase in mechano-responsive or osteogenic gene expression, and cortical bone formation in tibias of adult mice.

Radiation desiccation response motif-like sequences are involved in transcriptional activation of the Deinococcal ssb gene by ionizing radiation but not by desiccation.

Single-stranded-DNA binding protein (SSB) levels during poststress recovery of Deinococcus radiodurans were significantly enhanced by (60)Co gamma rays or mitomycin C treatment but not by exposure to UV rays, hydrogen peroxide (H2O2), or desiccation. Addition of rifampin prior to postirradiation recovery blocked such induction. In silico analysis of the ssb promoter region revealed a 17-bp palindromic radiation/desiccation response motif (RDRM1) at bp -114 to -98 and a somewhat similar sequence (RDRM2) at bp -213 to -197, upstream of the ssb open reading frame. Involvement of these cis elements in radiation-responsive ssb gene expression was assessed by constructing transcriptional fusions of edited versions of the ssb promoter region with a nonspecific acid phosphatase encoding reporter gene, phoN. Recombinant D. radiodurans strains carrying such constructs clearly revealed (i) transcriptional induction of the ssb promoter upon irradiation and mitomycin C treatment but not upon UV or H2O2 treatment and (ii) involvement of both RDRM-like sequences in such activation of SSB expression, in an additive manner.

Constrained tibial vibration does not produce an anabolic bone response in adult mice.

Osteoporosis is characterized by low bone mass and increased fracture risk. High frequency, low-amplitude whole-body vibration (WBV) has been proposed as a treatment for osteoporosis because it can stimulate new bone formation and prevent trabecular bone loss. We developed constrained tibial vibration (CTV) as a method for controlled vibrational loading of the lower leg of a mouse. We first subjected mice to five weeks of daily CTV loading (0.5 G maximum acceleration) with loading parameters chosen to independently investigate the effects of strain magnitude, loading frequency, and cyclic acceleration on the adaptive response to vibration. We hypothesized that mice subjected to the highest magnitude of dynamic strain would have the largest bone formation response. We observed a slight, local benefit of CTV loading on trabecular bone, as BV/TV was 5.2% higher in the loaded vs. non-loaded tibia of mice loaded with the highest bone strain magnitude. However, despite these positive differences, we observed significantly lower measures of trabecular structure in both loaded and non-loaded tibias from CTV loaded mice compared to Sham and Baseline Control animals, indicating a negative systemic effect of CTV on trabecular bone. Based on this evidence, we conducted a follow-up study wherein mice were subjected to CTV or sham loading, and tibias were scanned at the beginning and end of the study period using in vivo microCT. Consistent with the findings of the first study, trabecular BV/TV in both tibias of CTV loaded and Sham mice was, on average, 36% and 31% lower on day 36 than day 0, respectively, compared to 20% lower in Age-Matched Controls over the same time period. Contrary to the first study, there were no differences between loaded and non-loaded tibias in CTV loaded mice, providing no evidence for a local benefit of CTV. In summary, 5 weeks of daily CTV loading of mice was, at best, weakly anabolic for trabecular bone in the proximal tibia, while daily handling and exposure to anesthesia was associated with significant loss of trabecular and cortical bone. We conclude that direct vibrational loading of bone in anesthetized, adult mice is not anabolic.