First report of Phakopsora euvitis causing leaf rust disease on grapevine (Vitis labrusca L.) in India.

Grapevine (Vitis labrusca L.), a member of the family Vitaceae and native of North America, is grown as a table grape. During the survey for the grapevine diseases in May 2022, we noticed numerous yellow pustules of rust on lower side of leaves of 'Bangalore Bule' in Nandi village (13°22'59.7"N77°42'33.4"E), Chikkaballapur district of Karnataka state of India. The crop was at maturity stage and the rust disease severity was determined using the scale given by Angelotti et al. (2008), which was up to 10%. The disease symptoms were numerous small raised yellow pustules on the abaxial surface corresponding to adaxial surface chlorotic spots. In severe conditions, spots cover the entire leaf and defoliation occurs. Similar disease symptoms were reported by Ono (2000); Weinert et al. (2003); and Primiano et al. (2017). The pathogenicity test was performed on cuttings of 'Bangalore Bule' grapevine in a glasshouse at 25±1°C. The urediniospores were collected from diseased leaves using a brush, 3×104 ml-1 suspension in distilled water was used for inoculation on the abaxial surface of leaves. Control plants were sprayed with distilled water. The leaves developed symptoms in 15-17 days after inoculation, and the pathogen was confirmed by symptomatology and microscopic observation of urediniospores. Urediniospores were short-pedicellate, sessile, obovoid to obovoid-ellipsoid, and uniformly echinulate with 42.98-32.54 x 31.37-25.15 µm in size. The aecial stage of the Phakopsora has been reported on an alternate host, Meliosma simplicifolia (Hosagoudar 1988). As the internal transcribed spacer (ITS) region offers some utility in the molecular detection of the Phakopsora genus (Rush et al. 2019), the pathogen was confirmed by studying the different regions in the ITS such as ITS1, 5.8S rRNA, and ITS2. Total DNA was extracted from urediniospore mass using the Macherey-Nagel kit (Duren, Germany) by following the manufacturer's protocol. The quantity of isolated DNA was checked using an Qubit 3.0 fluorometer (Invitrogen) before being subjected to polymerase chain reaction (PCR) amplification in a thermocycler (Eppendorf-vapo.protect) using ITS1 and ITS4 primers (IDT, Singapore) targeting ITS1, 5.8S rRNA, and ITS2 regions, and the obtained amplicon (~700 bp) was purified using Macherey- Nagel Nucleospin gel and PCR clean-up kit (Duren, Germany) as per the manufacturer's protocol and sequenced by Sanger's dideoxy chain-termination method [ABI 3730 (48 capillaries) electrophoresis]. The sequence was edited in BioEdit (https://bioedit.software.informer.com/7.2/) and aligned in MUSCLE, and the phylogenetic tree was constructed in MEGA 11 using the neighbor-joining method by following the maximum likelihood criterion (Kumar et al. 2018). The sequence data was deposited at NCBI (accession number OP221661). The basic local alignment search tool (BLAST) search sequence of the isolate Nandi-KA in GenBank revealed 97.91% homology with sequence of Phakopsora sp. (accession number KC815548.1) and 96.87% with Phakopsora euvitis (accession number AB354790.1). Based on disease symptoms, fungal morphology, the pathogenicity test, and ITS sequence, the fungus was identified as Phakopsora euvitis, the pathogen causing grapevine leaf rust disease. Though there are similar disease symptoms observed on grapevine in India (EPPO 2016); the pathogen was not confirmed. To our knowledge, this is the first report of Phakopsora euvitis causing leaf rust disease in grapevine (V. labrusca) in India.

Molecular engineering of a cryptic epitope in Spike RBD improves manufacturability and neutralizing breadth against SARS-CoV-2 variants.

There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines.

Molecular engineering of a cryptic epitope in Spike RBD improves manufacturability and neutralizing breadth against SARS-CoV-2 variants

There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines.

SARS-CoV-2 receptor binding domain displayed on HBsAg virus-like particles elicits protective immunity in macaques.

Authorized vaccines against SARS-CoV-2 remain less available in low- and middle-income countries due to insufficient supply, high costs, and storage requirements. Global immunity could still benefit from new vaccines using widely available, safe adjuvants, such as alum and protein subunits, suited to low-cost production in existing manufacturing facilities. Here, a clinical-stage vaccine candidate comprising a SARS-CoV-2 receptor binding domain-hepatitis B surface antigen virus-like particle elicited protective immunity in cynomolgus macaques. Titers of neutralizing antibodies (>104) induced by this candidate were above the range of protection for other licensed vaccines in nonhuman primates. Including CpG 1018 did not significantly improve the immunological responses. Vaccinated animals challenged with SARS-CoV-2 showed reduced median viral loads in bronchoalveolar lavage (~3.4 log10) and nasal mucosa (~2.9 log10) versus sham controls. These data support the potential benefit of this design for a low-cost modular vaccine platform for SARS-CoV-2 and other variants of concern or betacoronaviruses.

Gaslighting in alleged assault ascertained as Munchausen syndrome transcending to malingering in highly counterintuitive self inflicted acid burns.

A factitious disorder leading to the self-infliction of highly counter-intuitive burns was diagnosed in a middle-aged female. The injuries were otherwise alleged to have been sustained by assault inflicted upon her by an unknown person. The case was diagnosed by medico-legal interpretation of injuries, in spite of a highly deceptive and concocted history by the patient and her husband. The entity was unique in being associated with magnificent primary, secondary and tertiary gains. The exploitation of the morbid sequel to malinger by the patient, and the involvement of the husband for the prolongation of the illness of his wife for financial gains as gaslighting was highly unusual. The self-infliction of injuries over hands is seen in factitious disorder. However, a combination of a guarded self-immersion of the hands and feet in a corrosive by an illiterate female, followed by malingering to earn livelihood is unprecedented in factitious disorders. The delayed presentation which required amputation of all the limbs to save the life of the patient is a glaring highlight of this case.

Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor-binding domain in engineered Komagataella phaffii.

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply ongoing demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor-binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5X by alleviating protein folding stress. Removal of methanol from the production process enabled to scale up to a 1200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii.

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply on-going demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii ( Pichia pastoris ). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol-induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5x by alleviating protein folding stress. Removal of methanol from the production process enabled scale up to a 1,200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply on-going demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol-induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5x by alleviating protein folding stress. Removal of methanol from the production process enabled scale up to a 1,200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

Coding-Complete Genome Sequences of NITMA1086 and NITMA1139, Two SARS-CoV-2 Isolates from Belagavi District, Karnataka State, India, Harboring the D614G Mutation.

We announce the coding-complete genome sequences of two isolates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from two coronavirus disease 2019 (COVID-19)-positive samples (RNA isolated from nasopharyngeal swabs) from Belagavi District, Karnataka State, India. Mutational analysis revealed the presence of the D614G substitution in both the isolates.

Canine parvovirus NS1 protein exhibits anti-tumor activity in a mouse mammary tumor model.

Many viral proteins have the ability to kill tumor cells specifically without harming the normal cells. These proteins, on ectopic expression, cause lysis or induction of apoptosis in the target tumor cells. Parvovirus NS1 is one of such proteins, which is known to kill high proliferating tumor cells. In the present study, we assessed the apoptosis inducing ability of canine parvovirus type 2 NS1 protein (CPV2.NS1) in vitro in 4T1 cells, and found it to cause significant cell death due to induction of apoptosis through intrinsic or mitochondrial pathway. Further, we also evaluated the oncolytic activity of CPV2.NS1 protein in a mouse mammary tumor model. The results suggested that CPV2.NS1 was able to inhibit the growth of 4T1 induced mouse mammary tumor as indicated by significantly reduced tumor volume, mitotic, AgNOR and PCNA indices. Further, inhibition of tumor growth was found to be because of induction of apoptosis in the tumor cells, which was evident by a significant increase in the number of TUNEL positive cells. Further, CPV2.NS1 was also able to stimulate the immune cells against the tumor antigens as indicated by the increased CD4+ and CD8+ counts in the blood of CVP2.NS1 treated mice. Further optimization of the delivery of NS1 protein and use of an adjuvant may further enhance its anti-tumor activity.

Canine parvovirus NS1 induced apoptosis involves mitochondria, accumulation of reactive oxygen species and activation of caspases.

The non-structural protein (NS1) of parvoviruses plays an important role in viral replication and is thought to be responsible for inducing cell death. However, the detailed mechanism and the pathways involved in canine parvovirus type 2 NS1 (CPV2.NS1) induced apoptosis are not yet known. In the present study, we report that expression of CPV2.NS1 in HeLa cells arrests cells in G1 phase of the cell cycle and the apoptosis is mitochondria mediated as indicated by mitochondrial depolarization, release of cytochrome-c and activation of caspase 9. Treatment of cells with caspase 9 inhibitor Z-LEHD-FMK reduced the induction of apoptosis significantly. We also report that expression of CPV2.NS1 causes accumulation of reactive oxygen species (ROS) and treatment with an antioxidant reduces the ROS levels and the extent of apoptosis. Our results provide an insight into the mechanism of CPV2.NS1 induced apoptosis, which might prove valuable in developing NS1 protein as an oncolytic agent.

Molecular cloning and characterization of arginine kinase gene of Toxocara canis.

Toxocara canis is an important gastrointestinal nematode of dogs and also a causative agent of visceral larva migrans in humans. Arginine kinase (AK) gene is one of the important biomolecule of phosphagen kinase of T. canis which is emerging as an exciting novel diagnostic target in toxocarosis. The present study was carried out to clone and characterize AK gene of T. canis for future utilization as a diagnostic molecule. Total RNA was extracted from intact adult worms and reverse transcription was done with oligo dT primers to obtain complementary DNA (cDNA). Polymerase chain reaction (PCR) was carried out using cDNA as template with specific primers which amplified a product of 1,202 bp. The amplicon was cloned into pDrive cloning vector and clone was confirmed by colony PCR and restriction endonuclease analysis. Sequence analysis of the gene showed 99.8 and 77.9 % homology with the published AK gene of T. canis (EF015466.1) and Ascaris suum respectively. Structural analysis shown that the mature AK protein consist of 400 amino acids with a molecular wt of 45360.73 Da. Further expression studies are required for producing the recombinant protein for its evaluation in the diagnosis of T. canis infection in humans as well as in adult dogs.

Canine parvovirus type 2a (CPV-2a)-induced apoptosis in MDCK involves both extrinsic and intrinsic pathways.

The canine parvovirus type 2 (CPV-2) causes an acute disease in dogs. It has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. In this paper, we evaluated the apoptotic potential of the "new CPV-2a" in MDCK cells and elucidated the mechanism of the induction of apoptosis. The exposure of MDCK cells to the virus was found to trigger apoptotic response. Apoptosis was confirmed by phosphatidylserine translocation, DNA fragmentation assays, and cell cycle analysis. Activation of caspases-3, -8, -9, and -12 and decrease in mitochondrial potential in CPV-2a-infected MDCK cells suggested that the CPV-2a-induced apoptosis is caspase dependent involving extrinsic, intrinsic, and endoplasmic reticulum pathways. Increase in p53 and Bax/Bcl2 ratio was also observed in CPV-2a-infected cells.

Exploring transplant opportunities in Hmong culture: a case report.

A clinical case is used to explore the ethical complexities of solid organ donation and transplantation within the Hmong community in the United States. Although many cultures can present various ethical issues, the challenges of the Hmong belief system are unique and distinctly complex. Ways for the medical team to integrate with the Hmong value system to attempt to create an environment of transcultural respect and appreciation are described.

Injuries to neck structures in deaths due to constriction of neck, with a special reference to hanging.

This prospective study aimed at examining various injuries to the neck structures in deaths due to constriction of neck. Neck dissection technique, as advocated by Prinsloo and Gordon was undertaken to study the injuries to the thyro-hyoid complex, strap muscles, carotid vessels, etc. Of the 1746 medico-legal autopsies, conducted during the study period, 5% were deaths due asphyxia of which 82% were those of constriction of neck. The 21-30 years age group accounted for the maximum number of cases (57%). Male:female ratio was 2:1. Hanging (69%) outnumbered other asphyxial deaths--ligature and/or manual strangulation, smothering, etc. Injury to the sternocleido-mastoid muscle (54%) was the commonest injury to the neck structures. The hyoid bone was fractured in 21% cases, while the thyroid cartilage was fractured in 17% cases. Complete hanging was noted in 68% of cases while the hanging was atypical in 88%. Fixed knot was found to have been used in 71%. A single loop round the neck was observed in 80% of the cases and it was above the level of thyroid in 58% cases. Most cases of the fracture of the laryngo-hyoid complex were in the 41-60 year age group, 72% and the fracture was on the same side as the knot in 52% cases. Majority used soft daily wear articles of clothing like a sari (32%) or chunni (24%). Asphyxial deaths due to constriction of neck being common in all parts of the world, prospective studies in different setups to examine the profile of neck structure injuries are needed so as to differentiate the suicidal or homicidal nature of such deaths with a greater certainty.

Analysis of results of surgical treatment of posttraumatic stiff elbow.

BACKGROUND: Surgical management of posttraumatic elbow stiffness has been reported with poor outcome following treatment. Sequential release in earlier stages of stiffness yielded much better results. The goal of our study was to assess the outcome in improvement of the range of motion of the elbow after surgical release and to analyze a tailor-made approach according to individual needs to yield good result. MATERIALS AND METHODS: A prospective study was conducted in 47 cases of elbow stiffness due to various types of injuries. All the cases were treated with sequential release if there was no progress after adequate supervised conservative management except in unreduced dislocations. All the cases were followed up for a minimum period of 24 months. Overall outcome was rated with the functional scoring system by Mayo Clinic Performance Index. RESULTS: Twenty-five (44.68%) out of 47 patients had excellent results with a mean preoperative range of motion of 33.9 degrees and postoperative range of motion of 105 degrees with net gain in range of motion of 71.1 degrees ('t' test value is 19.27, P < 0.01). None of the patients had elbow instability. Patients not having heterotopic ossification, who underwent surgery from three to six months post injury had a mean gain of 73.5 degrees. In patients who waited for more than six months had mean gain of 66.8 degrees. However, the results in cases having heterotopic ossification followed a slightly different pattern. In cases where release was performed from three months to six months had mean gain of 77.5 degrees. Cases in which release was performed after six months had gain of 57.1 degrees. CONCLUSIONS: In cases of posttraumatic elbow stiffness after a failed initial conservative treatment, early arthrolysis with sequential surgical soft tissue release yields good result than delayed surgery.

Catheter-directed therapy for DVT after pancreas transplantation.

INTRODUCTION: Iliac vein deep venous thrombosis (DVT) ipsilateral to the pancreas transplant can lead to severe leg edema and compromise graft function. Treatment modalities for iliac vein DVT in the pancreas transplant recipient are limited. METHODS: Medical records of patients receiving pancreas transplants at a single center from November 1989 to July 2003 were reviewed retrospectively, identifying patients with iliac vein DVT. There were 287 pancreas transplants performed during this time. Pancreas transplantation in all recipients was performed in the right iliac fossa with the arterial supply consisting of a donor iliac artery Y interposition graft. Systemic venous drainage was to the iliac vein. Exocrine drainage was enteric or to the bladder. RESULTS: Four (1.4%) cases of iliac DVT were identified. All patients manifested lower extremity edema ipsilateral to the pancreas transplant. DVT was detected by ultrasound on days 4, 5, 13, and 60 post-transplant. In all cases, the iliac vein caudad to the pancreatic venous anastomosis was noted to be stenotic. Management involved balloon dilatation and endovascular stent placement in one patient, thrombolysis with tissue plasma antigen (t-PA) followed by stent placement in one patient, and percutaneous mechanical thrombectomy in two patients. All patients had improvement in leg edema and two patients continue to have good pancreatic allograft function. CONCLUSIONS: Iliac DVT is a rare complication of pancreas transplantation that usually develops in an area of stenosis caudad to the pancreatic venous anastomosis. Catheter-based treatment modalities with use of endovascular stents for treatment of underlying stenoses can serve as an adjunct in treating these complications.

Septicemia as a cause of death in burns: an autopsy study.

In burn victims, invasion by the bacteria is not unexpected, despite advances in antibiotics, and it has been reported that in the absence of topical therapy, the superficial areas of burn wound contain up to 100 million organisms per gram of tissue within 48h following the injury. We examined the autopsy reports of 334 cases who died because of complicated burns and who underwent medico-legal autopsy during a period of 5 years to study the rate of infection/sepsis. It was observed that in 65% of fatal burn cases, septicemia was the cause of death. Pseudomonas aeuroginosa and Klebsiella sp. were the most common organism, isolated either singly or in combination in 29% and 28% cases, respectively. High mortality from burns in young married women has been recognized as an alarming and contentious problem in India, particularly among the low socio-economic groups. We found that the females aged 21-25 were the most common victims accounting for 37% of burn fatalities due to septicemia. It was concluded that to carry out periodic review of patterns of isolation and susceptibility profiles of microorganisms infecting burn wounds should be a routine in all burn units. In view of the limited resources of developing countries, we recommend the use of available scoring systems to estimate burn outcome so that the best care can be directed to those who have a better chance of improvement.

Neck structure injuries in Hanging--comparing retrospective and prospective studies.

Deaths due to hanging are common among suicides. Various studies in the forensic literature have reported considerable differences in the frequency of hyoid bone or thyroid cartilage fractures and injuries to the musculature and the vasculature of the neck. Some important reasons to which these variations could be attributed include: lack of a common method for examination of neck structures, varying degrees of thoroughness in examining the neck structures and lack of seriousness in the documentation of the findings (as cases of hanging are almost always suicidal) thus affecting the results of retrospective studies. The present study was conducted retrospectively (1994 to 1999) and prospectively (2000 to 2003) on 108 cases of hanging. Seventy-one per cent of the cases were male and 29% female, aged between 15 to 60 years. Hanging was typical in 20% of cases and atypical in 80%; it was complete hanging in 46% of cases and incomplete in 54% of cases. Neck structure fractures were found to be more common in atypical complete hangings; the incidence was found to be 10% in the retrospective group and 27% in the prospective group. Contusions and lacerations of musculature were found in 20% of cases in the retrospective group and 34% in the prospective group, thus stressing the need for thorough postmortem examinations and meticulous documentation of the findings.

Missed diagnoses in trauma patients vis-à-vis significance of autopsy.

Post-mortem examination is considered to be the gold standard for the critique of medical practice, providing a quality control tool for the retrospective evaluation of diagnoses and treatment. Performing autopsies also facilitates new insight about the pathogenesis of disease and effects of therapy, gives feedback to clinical research protocols, provides epidemiological information and occasionally helps to console and reassure grieving families that death was inevitable. Its significance becomes paramount in cases of missed diagnosis in trauma-related deaths. The true incidence of missed diagnoses in trauma-related deaths is unknown, because autopsy is conducted in only about 50% of injury-related deaths. Few studies have documented the frequency of missed diagnoses leading to deaths specifically in the trauma ICU population. The present study is an attempt to evaluate the incidence and nature of missed injuries and complications in trauma-related deaths given an autopsy rate of close to 100%. This study also sought to identify the primary factors contributing to each missed injury. However, the study is in no way intended to assigning blame to human or system errors. Rather, it is focussed specifically on the issue of whether autopsy can be useful to provide feedback in identifying clinical problems of trauma patients.