Insights into the multifunctional roles of geminivirus-encoded proteins in pathogenesis.

Geminiviruses are a major threat to agriculture in tropical and subtropical regions of the world. Geminiviruses have small genome with limited coding capacity. Despite this limitation, these viruses have mastered hijacking the host cellular metabolism for their survival. To compensate for the small size of their genome, geminiviruses encode multifunctional proteins. In addition, geminiviruses associate themselves with satellite DNA molecules which also encode proteins that support the virus in establishing successful infection. Geminiviral proteins recruit multiple host factors, suppress the host defense, and manipulate host metabolism to establish infection. We have updated the knowledge accumulated about the proteins of geminiviruses and their satellites in the context of pathogenesis in a single review. We also discuss their interactions with host factors to provide a mechanistic understanding of the infection process.

A geminivirus betasatellite damages the structural and functional integrity of chloroplasts leading to symptom formation and inhibition of photosynthesis.

Geminivirus infection often causes severe vein clearing symptoms in hosts. Recently a betasatellite has emerged as a key regulator of symptom induction. To understand the host-betasatellite interactions in the process of symptom development, a systematic study was carried out involving symptoms induced by a betasatellite associated with radish leaf curl disease (RaLCB) in Nicotiana benthamiana. It has been found that ßC1 protein localized to chloroplasts of host cells, and RaLCB lacking ßC1, which failed to produce symptoms, had no effect on chloroplast ultrastructure. Vein flecking induced by transiently expressed ßC1 was associated with chloroplast ultrastructure. In addition, the betasatellite down-regulates expression of genes involved in chlorophyll biosynthesis as well as genes involved in chloroplast development and plastid translocation. Interestingly, the expression of key host genes involved in chlorophyll degradation remains unaffected. Betasatellite infection drastically reduced the numbers of active reaction centres and the plastoquinol pool size in leaves exhibiting vein clearing symptoms. Betasatellite-mediated impediments at different stages of chloroplast functionality affect the photosynthetic efficiency of N. benthamiana. To the best of the authors' knowledge, this is the first evidence of a chloroplast-targeting protein encoded by a DNA virus which induces vein clearing and structurally and functionally damages chloroplasts in plants.

Closing the access barrier for effective anti-malarials in the private sector in rural Uganda: consortium for ACT private sector subsidy (CAPSS) pilot study.

BACKGROUND: Artemisinin-based combination therapy (ACT), the treatment of choice for uncomplicated falciparum malaria, is unaffordable and generally inaccessible in the private sector, the first port of call for most malaria treatment across rural Africa. Between August 2007 and May 2010, the Uganda Ministry of Health and the Medicines for Malaria Venture conducted the Consortium for ACT Private Sector Subsidy (CAPSS) pilot study to test whether access to ACT in the private sector could be improved through the provision of a high level supply chain subsidy. METHODS: Four intervention districts were purposefully selected to receive branded subsidized medicines - "ACT with a leaf", while the fifth district acted as the control. Baseline and evaluation outlet exit surveys and retail audits were conducted at licensed and unlicensed drug outlets in the intervention and control districts. A survey-adjusted, multivariate logistic regression model was used to analyse the intervention's impact on: ACT uptake and price; purchase of ACT within 24 hours of symptom onset; ACT availability and displacement of sub-optimal anti-malarial. RESULTS: At baseline, ACT accounted for less than 1% of anti-malarials purchased from licensed drug shops for children less than five years old. However, at evaluation, "ACT with a leaf" accounted for 69% of anti-malarial purchased in the interventions districts. Purchase of ACT within 24 hours of symptom onset for children under five years rose from 0.8% at baseline to 26.2% (95% CI: 23.2-29.2%) at evaluation in the intervention districts. In the control district, it rose modestly from 1.8% to 5.6% (95% CI: 4.0-7.3%). The odds of purchasing ACT within 24 hours in the intervention districts compared to the control was 0.46 (95% CI: 0.08-2.68, p=0.4) at baseline and significant increased to 6.11 (95% CI: 4.32-8.62, p<0.0001) at evaluation. Children less than five years of age had "ACT with a leaf" purchased for them more often than those aged above five years. There was no evidence of price gouging. CONCLUSIONS: These data demonstrate that a supply-side subsidy and an intensive communications campaign significantly increased the uptake and use of ACT in the private sector in Uganda.

Joint-sparing or physeal-sparing diaphyseal resections: the challenge of holding small fragments.

BACKGROUND: Joint-sparing or physeal-sparing diaphyseal resections are technically challenging when only a small length of bone is available for implant purchase. QUESTIONS/PURPOSES: We describe a series of cases with the aim of generating some guidelines as to the choice of reconstruction method and the implant used. METHODS: We retrospectively reviewed 25 patients with diaphyseal resections in which the remaining epiphyseal or metaphyseal segment provided 3 cm or less of purchase. Reconstruction was performed with bone (allograft, extracorporeally radiated autograft, or vascularized fibula) in 19 cases or a custom diaphyseal implant (CDI) in six. The implants used for holding the bone construct varied from standard plates to custom plates. The presence of union, function, complications, and disease status at last followup was recorded. RESULTS: Sixteen of the 25 patients are disease-free and alive with the original construct at a median followup of 34 months (range, 12-66 months). Implant-related complications such as plate breakage (four) and angulation (three) happened more frequently when weak plates such as reconstruction plates were used. Local recurrence with pulmonary metastases occurred in two cases. The two deep infections required an amputation or rotationplasty for control. Custom plates were successful in three of four patients. CONCLUSIONS: Weak plates such as reconstruction plates are best avoided for these reconstructions. Custom plates allow secure fixation with technical ease. CDIs allow immediate weightbearing and ability to lengthen with predictable good functional short-term outcome.

Management of severe crouch gait in children and adolescents with cerebral palsy.

BACKGROUND: Crouch gait in cerebral palsy is associated with spasticity and contracture of the hamstrings and weakness of the extensors of the hip and knee and ankle plantar flexors. Different treatment options have been described in the literature to deal with this difficult problem. We devised a different protocol of treatment aimed at correction of the flexion deformity of the knee, weakening of the hamstrings, and augmenting the power of the knee and hip extension, which we used on 17 children with severe crouch. METHODS: This surgery, performed in 2 stages, entailed shortening of the femur, plication of the patellar tendon, transfer of the semitendinous to the back of the femur, and fractional lengthening of the other hamstrings. The degree of fixed deformity, the popliteal angle, quadriceps power, range of knee motion, ambulatory status and the efficiency of gait, and the position of the patella were evaluated before surgery and again after a minimum 2-year follow-up. RESULTS: The gait improved and the power of the quadriceps and the range of knee motion increased. The flexion deformity and popliteal angle decreased significantly. Patella alta was corrected and all fragmentation of the tibial tuberosity and fractures of the patella healed. The Functional Mobility Scores and the ambulatory capacity increased in all the children. There was no evidence of damage to the sciatic nerve in any patient. CONCLUSIONS: The method of treatment of severe crouch gait outlined in this study seems to be an effective and safe method of dealing with this difficult problem. LEVEL OF EVIDENCE: IV.

Surgical Management of Bone Sarcomas with an Inappropriate Biopsy Site: A Case Series and Review of Literature.

AIM: To study bone sarcoma patients who underwent limb salvage surgery with an inappropriate biopsy site and their surgical management. MATERIALS AND METHODS: There were 28 patients with bone sarcoma who underwent limb salvage surgery from 2015 - 2019, among them 4 patients had inappropriate biopsy scar. They were analyzed with respect to modification of incision, extent of flap elevation, difficulties in exposure of neurovascular structures, reconstructive methods and post-operative wound morbidity. RESULTS: In all cases biopsy was done in outside hospital. One patient had open biopsy, remaining all were core needle biopsies. Out of the 4, three patients had distal femur lesion and one patient had proximal tibial lesion. Patient with proximal tibial lesion had biopsy site placed anteriorly over tibia. Patient underwent proximal tibial resection with anteromedial incision. Remaining three patients had distal femur lesion. One patient had pathological fracture with internal fixation done with lateral incision, and biopsy site placed medially. During surgery lateral incision was taken to include internal fixation scar with the distal end curved medially and extended over leg after including the biopsy site. One patient had distal femur lesion with laterally placed biopsy site, surgery done with lateral approach and medial gastrocnemius flap used to cover the prosthesis. One patient had anteriorly placed biopsy site, so after including the inappropriate biopsy site the lateral incision was curved medially and extended over leg. Two out of these four patients had skin necrosis; one patient had partial thickness skin necrosis and the other full thickness skin necrosis which was managed conservatively. CONCLUSION: In case of limb salvage surgery, skin incisions should be planned taking extent of lesion and biopsy scars into account. Good knowledge of vascular supply and incisions required if and when surgery is planned helps in selecting proper site for biopsy.

Memory protective effect of indomethacin against electroconvulsive shock-induced retrograde amnesia in rats.

BACKGROUND: Nonsteroidal antiinflammatory drugs (NSAIDs) have been suggested to retard cognitive decrements in patients with Alzheimer's disease. We postulated that NSAIDs also may protect acute disruption of memory. METHODS: We studied the effect of indomethacin (4 mg/kg/day) administered daily for 19 days on retrograde amnesia induced by two once-daily electroconvulsive shocks in rats. RESULTS: Indomethacin produced statistically significant prolongation of recall latency in a passive avoidance task using a step-down apparatus. CONCLUSIONS: Our study suggests that NSAIDs may prevent memory disruption through other mechanisms apart from attenuating chronic inflammation. In patients receiving electroconvulsive therapy, as in those diagnosed with Alzheimer's disease, antiinflammatory drugs may hold promise in the attenuation of cognitive impairments.

Keratocyte loss in corneal infection through apoptosis: a histologic study of 59 cases.

BACKGROUND: Keratocyte loss by apoptosis following epithelial debridement is a well-recognized entity. In a study of corneal buttons obtained from patients of corneal ulcer undergoing therapeutic keratoplasty, we observed loss of keratocytes in the normal appearing corneal stroma, surrounding the zone of inflammation. Based on these observations, we hypothesized that the cell loss in the inflammatory free zone of corneal stroma is by apoptosis that could possibly be a non-specific host response, independent of the nature of infectious agent. METHODS: To test our hypothesis, in this study, we performed Terminal deoxyribonucleotidyl transferase-mediated d-Uridine 5" triphosphate Nick End Labelling (TUNEL) staining on 59 corneal buttons from patients diagnosed as bacterial, fungal, viral and Acanthamoeba keratitis. The corneal sections were reviewed for morphologic changes in the epithelium, stroma, type, degree and depth of inflammation, loss of keratocytes in the surrounding stroma (posterior or peripheral). TUNEL positivity was evaluated in the corneal sections, both in the zone of inflammation as well as the surrounding stroma. A correlation was attempted between the keratocyte loss, histologic, microbiologic and clinical features. RESULTS: The corneal tissues were from 59 patients aged between 16 years and 85 years (mean 46 years) and included fungal (22), viral (15), bacterial (14) and Acanthamoeba (8) keratitis. The morphological changes in corneal tissues noted were: epithelial ulceration (52, 88.1%), destruction of Bowman's layer (58, 99%), mild to moderate (28; 47.5%) to severe inflammation (31; 52.5%). Morphologic evidence of disappearance or reduced number of keratocytic nuclei in the corneal stroma was noted in 49 (83%) cases; while the TUNEL positive brown cells were identified in all cases 53/54 (98%), including cases of fungal (19), bacterial (14), viral (13), and Acanthamoeba keratitis. TUNEL staining was located mostly in the deeper stroma and in few cases the peripheral stroma. TUNEL positivity was also noted with the polymorphonuclear infiltrates and in few epithelial cells (10 of 59, 17%) cases, more with viral infections (6/10; 60%). CONCLUSIONS: We report apoptotic cell death of keratocytes in the corneal stroma in infectious keratitis, a phenomenon independent of type of infectious agent. The inflammatory cells in the zone of inflammation also show evidence of apoptotic cell death. It could be speculated that the infective process possibly triggers keratocyte loss of the surrounding stroma by apoptosis, which could possibly be a protective phenomenon. It also suggests that necrotic cell death and apoptotic cell deaths could occur simultaneously in infective conditions of the cornea.

"Metabolic signature" of the lesions on F18-fluoro-deoxyglucose positron emission tomography: A clue to the underlying pathology.

Increase in glycolytic pathway, forms one of the major adaptations in various cancer types. This can be imaged using (18)F-fluoro-deoxyglucose positron emission tomography/computed tomography (FDG PET). The intensity of FDG avidity is an indirect marker of the grade of the tumor. We present a case where FDG PET demonstrated a known chondrosarcoma and two other incidental lesions. The intensity of avidity in each of the lesions was grossly incongruent from the chondrosarcoma and further investigation proved the lesions to be two distinct primary malignancies, pathologically different from the known chondrosarcoma. We present the case to highlight the fact that the grade of FDG avidity is a clue to the pathological nature of the lesion and should always be considered while interpreting PET images.

Alkylidene Oxapenem ß-Lactamase Inhibitors Revisited: Potent Broad Spectrum Activity but New Stability Challenges.

We present a comprehensive study of C6-alkylidene containing oxapenems. We show that this class of ß-lactamase inhibitors possesses an unprecedented spectrum with activity against class A, C, and D enzymes. Surprisingly, this class of compounds displayed significant photolytic instability in addition to the known hydrolytic instability. Quantum mechanical calculations were used to develop models to predict the stability of new analogues.

A multi-site validation in India of the line probe assay for the rapid diagnosis of multi-drug resistant tuberculosis directly from sputum specimens.

UNLABELLED: Rifampicin (R) and isoniazid (H) are key first-line anti-tuberculosis drugs. Failure to detect resistance to these two drugs early results in treatment failure and poor clinical outcomes. The study purpose was to validate the use of the GenoType MTBDRplus line probe assay (LPA) to detect resistance to R and H in Mycobacterium tuberculosis strains directly from smear-positive sputum samples in India. METHOD: Smear positive sputum specimens from 320 patients were subjected to LPA and results compared against those from conventional Lowenstein Jensen (LJ) culture and drug susceptibility testing (C&DST). All specimens with discordant R DST results were subjected to either sequencing of the rpoB gene and/or repeat DST on liquid culture (MGIT 960) at a National Reference Laboratory. RESULTS: Significantly higher proportion of interpretable results were observed with LPA compared to LJ C&DST (94% vs. 80%, p-value <0.01). A total of 248 patients had both LJ and LPA DST results available; 232 (93.5%) had concordant R DST results. Among the 16 discordant R DST results, 13 (81%) were resolved in agreement with LPA results. Final LPA performance characteristics were sensitivity 96% (CI: 90%-98%), specificity 99% (CI: 95%-99%), positive predictive value 99% (CI: 95%-99%), and negative predictive value 95% (CI: 89%-98%). The median turnaround testing time, including specimen transportation time, on LPA was 11 days as compared with 89 days for LJ C&DST. CONCLUSIONS: LPA proved highly accurate in the rapid detection of R resistance. The reduction in time to diagnosis may potentially enable earlier commencement of the appropriate drug therapy, leading to some reduction of transmission of drug-resistant strains.

Operational challenges in diagnosing multi-drug resistant TB and initiating treatment in Andhra Pradesh, India.

BACKGROUND: Revised National TB Control Programme (RNTCP), Andhra Pradesh, India. There is limited information on whether MDR-TB suspects are identified, undergo diagnostic assessment and are initiated on treatment according to the programme guidelines. OBJECTIVES: To assess i) using the programme definition, the number and proportion of MDR-TB suspects in a large cohort of TB patients on first-line treatment under RNTCP ii) the proportion of these MDR-TB suspects who underwent diagnosis for MDR-TB and iii) the number and proportion of those diagnosed as MDR-TB who were successfully initiated on treatment. METHODS: A retrospective cohort analysis, by reviewing RNTCP records and reports, was conducted in four districts of Andhra Pradesh, India, among patients registered for first line treatment during October 2008 to December 2009. RESULTS: Among 23,999 TB patients registered for treatment there were 559 (2%) MDR-TB suspects (according to programme definition) of which 307 (55%) underwent diagnosis and amongst these 169 (55%) were found to be MDR-TB. Of the MDR-TB patients, 112 (66%) were successfully initiated on treatment. Amongst those eligible for MDR-TB services, significant proportions are lost during the diagnostic and treatment initiation pathway due to a variety of operational challenges. The programme needs to urgently address these challenges for effective delivery and utilisation of the MDR-TB services.

How do patients who fail first-line TB treatment but who are not placed on an MDR-TB regimen fare in South India?

SETTING: Seven districts in Andhra Pradesh, South India. OBJECTIVES: To a) determine treatment outcomes of patients who fail first line anti-TB treatment and are not placed on an multi-drug resistant TB (MDR-TB) regimen, and b) relate the treatment outcomes to culture and drug susceptibility patterns (C&DST). DESIGN: Retrospective cohort study using routine programme data and Mycobacterium TB Culture C&DST between July 2008 and December 2009. RESULTS: There were 202 individuals given a re-treatment regimen and included in the study. Overall treatment outcomes were: 68 (34%) with treatment success, 84 (42%) failed, 36 (18%) died, 13 (6.5%) defaulted and 1 transferred out. Treatment success for category I and II failures was low at 37%. In those with positive cultures, 81 had pan-sensitive strains with 31 (38%) showing treatment success, while 61 had drug-resistance strains with 9 (15%) showing treatment success. In 58 patients with negative cultures, 28 (48%) showed treatment success. CONCLUSION: Treatment outcomes of patients who fail a first-line anti-TB treatment and who are not placed on an MDR-TB regimen are unacceptably poor. The worst outcomes are seen among category II failures and those with negative cultures or drug-resistance. There are important programmatic implications which need to be addressed.

Perinuclear localization of slow troponin C m RNA in muscle cells is controlled by a cis-element located at its 3' untranslated region.

The process of mRNA localization within a specific cytoplasmic region is an integral aspect of the regulation of gene expression. Furthermore, colocalization of mRNAs and their respective translation products may facilitate the proper assembly of multi-subunit complexes like the thick and thin filaments of muscle. This postulate was tested by investigating the cytoplasmic localization of three mRNAs-the alpha-actin, slow troponin C (sTnC), and slow troponin I (sTnI), which encode different poly-peptide partners of the thin filament. Using in situ hybridization we showed that all three thin filament mRNAs are localized in the perinuclear cytoplasm of cultured C2C12 muscle cells. Their localization differs from that of the nonmuscle beta-actin mRNA, which is localized in the peripheral region of both proliferating nondifferentiated myoblasts and the differentiated myocytes. Analysis of the localization signal of the sTnC mRNA showed that a 40-nucleotide-long region of the sTnC mRNA 3' UTR is sufficient to confer the perinuclear localization on a heterologous reporter beta-Gal mRNA. This localization signal showed tissue specificity and worked only in the differentiated myocytes, but not in the proliferating myoblasts or in HeLa cells. The predicted secondary structure of the localization signal suggests the presence of multiple stem and loop structures in this region of the 3' UTR. Mutations within the stem region of the localization signal, which abolish the base pairing in this region, significantly reduced its perinuclear mRNA localization activity. Using UV-induced photo-cross-linking of RNA and proteins we found that a myotube-specific 42-kDa polypeptide binds to the localization signal.