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Background: Plasmid-mediated resistance to the last-resort drugs: carbapenems and colistin is an emerging public health threat. The studies on the prevalence and co-expression of resistant genes among livestock and human pathogens are rare in Nepal. This is the first study in Nepal exploring the prevalence and co-existence of colistin resistance gene, mcr -1 along with carbapenemase resistance gene, OXA -48 in Escherichia coli isolated from poultry and clinical specimens., Methods: A total of 240 rectal swabs from chickens of five different poultry farms of Kathmandu valley and 705 mid-stream urine samples from human subjects attending Kantipur Hospital, Kathmandu were collected between August, 2018 and March, 2019. Rectal swabs and urine specimens were cultured. E. coli isolated from the specimens were screened for antimicrobial susceptibility testing (AST) using disk diffusion method'. Minimum inhibitory concentration (MIC) of colistin was determined by agar dilution method using 0.5 µg/ml to 32 µg/ml. The E. coli isolates were first screened for mcr - 1 followed by screening for OXA - 48 genes using conventional Polymerase chain reaction (PCR)., Results: Of the total samples analyzed, E. coli was isolated from 31.7% (76/240) of poultry and 7.9% (56/705) of clinical specimens. In AST, 80% (61/76) of E. coli from poultry and 79% (44/56) from clinical specimens were MDR. The phenotypic prevalence of colistin resistance in poultry specimens were 31.6% (24/76) and clinical specimens were 21.4% (12/56). In PCR assay, 27.6% (21/76) of poultry and 19.6% (11/56) of clinical isolates had colistin resistant mcr - 1 gene. MICs value of E. coli isolates ranged from 4 to 32 (µg/ml) in both clinical and poultry isolates. Prevalence of co-existing carbapenem resistance gene, OXA -48, among colistin resistant mcr -1 positive isolates was 38% (8/21) in poultry specimens and 18.2% (2/11) in clinical specimens., Conclusions: The high prevalence of colistin and carbapenem resistant genes, and their co-existence in plasmid DNA of E. coli isolates in this study suggests the possible spread to other animal, human and environmental pathogens. Molecular methods in addition to the conventional diagnostics in laboratories can help in early diagnosis, effective management and control of their potential transmission., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Introduction: Emergence and spread of antimicrobial resistance (AMR) is a global threat and significantly affects the treatment options for common infectious diseases. Inappropriate use of antibiotics, particularly third-generation cephalosporins, has contributed to the development of AMR. This study aims to determine the prevalence of extended-spectrum β-lactamase (ESBL) production in Escherichia coli and Klebsiella species isolated from various clinical samples., Methods: This cross-sectional study was conducted at International Friendship Children's Hospital, Kathmandu, Nepal, from August 2017 to January 2018. A total of 1443 samples that included urine, pus, wound swab, endotracheal tip, catheter tip, and blood were collected from pediatric patients below 15 years and processed by standard microbiological methods. Following sufficient incubation, isolates were identified by colony morphology, gram staining, and necessary biochemical tests. Identified bacterial isolates were then tested for antibiotic susceptibility test by modified Kirby-Bauer disk diffusion method and were subjected to ESBL screening by using 30 µg cefotaxime and ceftazidime. The ESBL production was confirmed by combination disk method., Results: From a total of 103 nonduplicated clinical isolates, E. coli (n = 79), Klebsiella pneumoniae (n = 18), and Klebsiella oxytoca (n = 6) were isolated from different clinical specimens. Of which, 64 (62.1%) exhibited multidrug resistance, and 29 (28.2%) were ESBL producers. All ESBL-producing isolates were resistant toward ampicillin, cefotaxime, ceftriaxone, and ceftazidime. Most ESBL producers were susceptible toward imipenem (89.7%; 26/29), nitrofurantoin (82.8%; 24/29), piperacillin/tazobactam (79.3%; 23/29), and amikacin (72.4%; 21/29)., Conclusions: A high prevalence of multidrug-resistant ESBL organisms was found in this study among pediatric patients. Treatment based on their routine identification and susceptibility to specific antibiotics is critical to halt the spread of AMR and ESBL., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)

Suicide is a major public health issue in Japan, with very high rates of death compared to other countries in the Asia Pacific. Foreigners living in Japan may be at increased risk of suicide, but little is known about how their risk of suicide differs from that of their country of origin or Japanese nationals. We used data on suicide mortality from the Japan Vital Registration System for the period 2012-2016 to analyze risk of suicide mortality in Japan for Japanese, Korean, Chinese, and other nationalities living in Japan, adjusting for age and separately by sex. We estimated standardized mortality rates using both the Japanese population as a reference, and also the population of the home nation of the foreign residents. We found that Korean nationals living in Japan have significantly higher mortality rates than Japanese nationals, and that the suicide mortality rate of Korean nationals living in Japan is higher than in their home country, but that this is not the case for Chinese or other nationals resident in Japan. Koreans living in Japan have a very high risk of mortality due to suicide which may reflect the special social, economic, and cultural pressures they face as a marginalized population in Japan.

Suicide is a major public health concern in Japan. This study aimed to characterize the trends in suicide mortality in Japan by method since 1979. Using data from the Japan vital registration system, we calculated age-standardized rates of suicide mortality separately by sex and method. We conducted a log-linear regression of suicide mortality rates separately by sex, and linear regression analysis of the proportion of deaths due to hanging, including a test for change in level and trend in 1998. While crude suicide rates were static over the time period, age-adjusted rates declined. The significant increase in suicide mortality in 1998 was primarily driven by large changes in the rate of hanging, with suicide deaths after 1998 having 36.7% higher odds of being due to hanging for men (95% CI: 16.3-60.8%), and 21.9% higher odds of being due to hanging for women (95% CI: 9.2-35.9%). Hanging has become an increasingly important method for committing suicide over the past 40 years, and although suicide rates have been declining continuously over this time, more effort is needed to prevent hanging and address the potential cultural drivers of suicide if the rate is to continue to decline in the future.

Voretigene neparvovec-rzyl was recently approved for the treatment of Leber congenital amaurosis, and the use of gene therapy for eye disease is attracting even greater interest. The eye has immune privileged status, is easily accessible, requires a reduced dosage of therapy due to its size and is highly compartmentalized, significantly reducing systemic spread. Adeno-associated virus (AAV), with its low pathogenicity, prolonged expression profile and ability to transduce multiple cell types, has become the leading gene therapy vector. Target diseases have moved beyond currently untreatable inherited dystrophies to common, partially treatable acquired conditions such as exudative age-related macular degeneration and glaucoma, but use of the technology in these conditions imposes added obligations for caution in vector design. This review discusses the current status of AAV gene therapy trials in genetic and acquired ocular diseases, and explores new scientific developments, which could help ensure effective and safe use of the therapy in the future., (© 2018 Royal Australian and New Zealand College of Ophthalmologists.)

Introduction: This study reviews the evidence for the use of Bortezomib (BZB), a first-in-class proteasome inhibitor in advanced Hepatocellular carcinoma (HCC). This review aims to delineate the role of BZB within the management of non-surgical and metastatic HCC, either as an alternative or as an adjunct to the current treatment paradigm., Areas Covered: In addition to BZB pharmacology and mechanism of action, safety and tolerance profiles of the drug obtained from clinical trials are explored. The utility of BZB as a therapeutic agent either alone or in combination with other therapies against HCC, including its application in both preclinical and clinical settings has been reviewed. In particular, we highlight the importance of preclinical evaluation of BZB as a combinatorial agent in synergism with other therapies for the use in the management of HCC., Expert Opinion: There has been much interest surrounding the use of BZB, a first-in-class proteasome inhibitor for HCC therapy. The discernment of outcomes of BZB clinical trials for HCC need to take into consideration the disease-specific factors that can affect survival outcomes including patient selection and aetiological differences. Further preclinical testing of BZB in combination with other therapeutic modalities can be important for eliciting enhanced anti-HCC effects.

A gene therapeutic platform needs to be both efficient and safe. The criterion of safety is particularly important for diseases like hepatocellular carcinoma (HCC), which develop in a background of an already compromised liver. Gene vectors can be constructed either by targeting HCC or by detargeting liver and/or other major organs. miRNA-based negative detargeting has gained considerable attention in recent times due to its effectiveness and the ease with which it can be adapted into current gene delivery vectors. In this study, we provide a proof-of-concept using miRNA199a as a negative targeting agent. We introduced vectors harboring reporters with miRNA199a binding sites in cells expressing high endogenous levels of miRNA199a and compared the reporter expression in HCC cells with low endogenous miRNA199a. We observed that the expression of reporters with miRNA199a binding sites is significantly inhibited in miRNA199a-positive cells, whereas minimal effect was observed in miRNA199a-negative HCC cells. In addition, we created a post-transcriptionally regulated suicide gene therapeutic system based on cytosine deaminase (CD)/5-fluorocytosine (5-FC) exploiting miRNA199a binding sites and observed significantly lower cell death for miRNA199a-positive cells. Furthermore, we observed a decrease in the levels of miRNA199 in 3D tumorspheres of miRNA199a-positive Hepa1-6 cells and a reduction in the inhibition of reporter expression after transfection in these 3D models when compared with 2D Hepa1-6 cells. In summary, we provide evidence of miRNA199a-based post-transcriptional detargeting with relevance to HCC gene therapy., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

In hepatocellular carcinoma (HCC), which usually develops in a cirrhotic liver, treatments preserving normal liver function and viability are vitally important. Here, we utilise the differential expression of miRNAs 122a and 199a between normal hepatocytes and HCC to generate vectors harbouring their binding sites for hepatocyte detargeting. Using a reporter gene, we observed a synergistic detargeting of cells expressing both miRNAs as well as cells expressing either of the miRNAs; while expression was retained in HCC cells negative for both miRNA122a and miRNA199a. Mimics and inhibitors for individual miRNAs were used to confirm these results. Furthermore, suicide gene therapy with cytosine deaminase (CD)/5-fluorocytosine system resulted in limited killing of cells expressing either of the two miRNAs. Finally, we report feasibility of using adeno associated virus (AAV) based vectors for delivery of this dual regulated gene delivery system. These results present a novel dual targeted system whereby miRNA122a and miRNA199a act either synergistically or independently in regulating transgene expression with vectors harbouring binding sites of both miRNAs and have implications in detargeting vectors from multiple cell types in the liver.

The global burden of hepatocellular carcinoma (HCC), one of the frequent causes of cancer-related deaths worldwide, is rapidly increasing partly due to the limited treatment options available for this disease and recurrence due to therapy resistance. Immune checkpoint inhibitors that are proved to be beneficial in the treatment of advanced melanoma and other cancer types are currently in clinical trials in HCC. These ongoing trials are testing the efficacy and safety of a few select checkpoints in HCC. Similar to observations in other cancers, these immune checkpoint blockade treatments as monotherapy may benefit only a fraction of HCC patients. Studies that assess the prevalence and distribution of other immune checkpoints/modulatory molecules in HCC have been limited. Moreover, robust predictors to identify which HCC patients will respond to immunotherapy are currently lacking. The objective of this study is to perform a comprehensive evaluation on different immune modulators as predictive biomarkers to monitor HCC patients at high risk for poor prognosis. We screened publically available HCC patient databases for the expression of previously well described immune checkpoint regulators and evaluated the usefulness of these immune modulators to predict high risk, patient overall survival and recurrence. We also identified the immune modulators that synergized with known immune evasion molecules programmed death receptor ligand-1 (PD-L1), programmed cell death protein-1 (PD-1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and correlated with worse patient outcomes. We evaluated the association between the expression of epithelial-to-mesenchymal transition (EMT) markers and PD-L1 in HCC patient tumors. We also examined the relationship of tumor mutational burden with HCC patient survival. Notably, expression of immune modulators B7-H4, PD-L2, TIM-3 , and VISTA were independently associated with worse prognosis, while B7-H4, CD73 , and VISTA predicted low recurrence-free survival. Moreover, the prognosis of patients expressing high PD-L1 with high B7-H4, TIM-3, VISTA, CD73 , and PD-L2 expression was significantly worse. Interestingly, PD-L1 expression in HCC patients in the high-risk group was closely associated with EMT marker expression and prognosticates poor survival. In HCC patients, high tumor mutational burden (TMB) predicted worse patient outcomes than those with low TMB.

Targeted gene delivery relies on the ability to limit the expression of a transgene within a defined cell/tissue population. MicroRNAs represent a class of highly powerful and effective regulators of gene expression that act by binding to a specific sequence present in the corresponding messenger RNA. Involved in almost every aspect of cellular function, many miRNAs have been discovered with expression patterns specific to developmental stage, lineage, cell-type, or disease stage. Exploiting the binding sites of these miRNAs allows for construction of targeted gene delivery platforms with a diverse range of applications. Here, we summarize studies that have utilized miRNA-regulated systems to achieve targeted gene delivery for both research and therapeutic purposes. Additionally, we identify criteria that are important for the effectiveness of a particular miRNA for such applications and we also discuss factors that have to be taken into consideration when designing miRNA-regulated expression cassettes.

In this study, we report a miRNA122a based targeted gene therapy for hepatocellular cancer stem cells (CSCs). First, we assessed the levels of miRNA122a in normal human hepatocytes, a panel of hepatocellular carcinoma (HCC) cell lines and hepatocellular CSCs observing its significant downregulation in HCC and CSCs. The miRNA122a binding site was then incorporated at the 3'-UTR of reporter genes gaussia luciferase (GLuc) and eGFP which resulted in significant hepatocyte detargeting. Using this strategy for the delivery of gene directed enzyme prodrug therapy (GDEPT) utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, we showed significant killing in cells with low or no miRNA122a while those cells, such as hepatocytes with high miRNA122a were largely spared. Next, we showed that CSC enriched tumorspheres exhibit a significant downregulation of miRNA122a expression providing a rational to exploit its binding site for targeted gene delivery. Using plasmids harboring reporters GLuc and eGFP with or without miR122a binding sites, we showed high reporter expression in the CSCs and little reported expression in the non-enriched cultures. Finally, we demonstrate the efficacy of miRNA122a based post-transcriptionally targeted GDEPT for hepatocellular CSCs., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to declare.

Hepatocellular carcinoma (HCC) is a major health problem as evidenced by its increasing incidence and high morbidity and mortality rates. Most patients with HCC have underlying liver disease and dysfunction which limits the current therapeutic options. Treatments that spare the liver and destroy the HCC are needed. Targeting transcriptional differences between HCC and liver cells may provide this therapeutic window. In this study, we examine the potential of the Glypican 3 (GPC3) promoter as a targeting strategy. GPC3 is an oncofetal protein belonging to the proteoglycan family which is normally only expressed during fetal development. However, in HCC, the expression of this protein is reactivated. Here, we show that GPC3 is expressed primarily in HCC and not in normal liver lines. We show that the GPC3 promoter can be used to drive expression of significantly more luciferase and eYFP in HCC cell lines compared to normal liver cells. Further, we show that vectors containing cytosine deaminase (CD) under GPC3 promotor control induced significantly more killing of HCC cell lines after treatment with 5-FC compared to normal liver cell lines. These data suggest that transcriptionally targeted delivery of transgene in HCC cells can be achieved using the GPC3 promoter and this targeting strategy produces limited toxicity to normal liver cells.

We studied the systematics of the subfamily Limenitidinae (Lepidoptera: Nymphalidae) using molecular methods to reconstruct a robust phylogenetic hypothesis. The molecular data matrix comprised 205 Limenitidinae species, four outgroups, and 11,327 aligned nucleotide sites using up to 18 genes per species of which seven genes (CycY, Exp1, Nex9, PolII, ProSup, PSb and UDPG6DH) have not previously been used in phylogenetic studies. We recovered the monophyly of the subfamily Limenitidinae and seven higher clades corresponding to four traditional tribes Parthenini, Adoliadini, Neptini, Limenitidini as well as three additional independent lineages. One contains the genera Harma + Cymothoe and likely a third, Bhagadatta , and the other two independent lineages lead to Pseudoneptis and to Pseudacraea . These independent lineages are circumscribed as new tribes. Parthenini was recovered as sister to rest of Limenitidinae, but the relationships of the remaining six lineages were ambiguous. A number of genera were found to be non-monophyletic, with Pantoporia , Euthalia, Athyma , and Parasarpa being polyphyletic, whereas Limenitis , Neptis , Bebearia , Euryphura, and Adelpha were paraphyletic., Competing Interests: The authors declare there are no competing interests.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer with high incidence globally. Increasing mortality and morbidity rates combined with limited treatment options available for advanced HCC press for novel and effective treatment modalities. Gene therapy represents one of the most promising therapeutic options. With the recent approval of herpes simplex virus for advanced melanoma, the field of gene therapy has received a major boost. Adeno-associated virus (AAV) is among the most widely used and effective viral vectors today with safety and efficacy demonstrated in a number of human clinical trials. This review identifies the obstacles for effective AAV based gene delivery to HCC which primarily include host immune responses and off-target effects. These drawbacks could be more pronounced for HCC because of the underlying liver dysfunction in most of the patients. We discuss approaches that could be adopted to tackle these shortcomings and manufacture HCC-targeted vectors. The combination of transductional targeting by modifying the vector capsid and transcriptional targeting using HCC-specific promoters has the potential to produce vectors which can specifically seek HCC and deliver therapeutic gene without significant side effects. Finally, the identification of novel HCC-specific ligands and promoters should facilitate and expedite this process.

Aim: To establish a model to enrich and characterize stem-like cells from murine normal liver and hepatocellular carcinoma (HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition (EMT)., Methods: In this study, we utilized a stem cell conditioned serum-free medium to enrich stem-like cells from mouse HCC and normal liver cell lines, Hepa 1-6 and AML12, respectively. We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating the RNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR (qRT-PCR). Next, we examined the relationship between stem cells and EMT using qRT-PCR., Results: Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor and heparin sulfate. The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell (CSC) marker Cd44 compared to parental cells grown as adherent cultures. We report that epithelial markers E-cadherin and ZO-1 were downregulated, while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres. The 3-dimensional spheres also exhibited changes in expression of Snai , Zeb and Twist family of EMT transcription factors., Conclusion: Our novel method successfully enriched stem-like cells which possessed an EMT phenotype. The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC., Competing Interests: Conflict-of-interest statement: The authors declare no conflict of interest. The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the United States Government. Brian J Morrison is a military service member. This work was prepared as part of his official duties. Title 17 U.S.C. §105 provides that Copyright protection under this title is not available for any work of the United States Government. Title 17 U.S.C. §101 defines a United States Government work as a work prepared by a military service member or employee of the United States Government as part of that person’s official duties.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide and its incidence is steadily rising. Currently, sorafenib remains the only approved standard treatment for patients with advanced HCC, as it has proven to increase survival in these patients. However, clinical and preclinical observations indicate that sorafenib treatment may have limited efficacy due to tumor progression from the rapid development of acquired resistance. Elucidation of the underlying mechanisms of evasive resistance to sorafenib is a major challenge in HCC research. In recent years, the role of epithelial-to-mesenchymal transition (EMT) in the advancement of HCC and development of drug resistance has gained increasing attention. EMT is a developmental multistep molecular and cellular reprogramming process that is hijacked by cancer cells to enable aggressiveness. In this review, we provide an overview of the currently available preclinical studies on the EMT mechanisms underlying resistance to sorafenib treatment. Recent studies report enrichment of cancer stem cells (CSCs) after sorafenib treatment. Interestingly, EMT process has been implicated in the generation of CSCs associated with therapy resistance. We discuss how combination of sorafenib with EMT inhibitors could enhance the clinical response to sorafenib, resulting in longer duration of responses, than observed with sorafenib monotherapy. In particular, we discuss how these new insights may facilitate rational development of combination therapies in the future to impact survival of patients with advanced HCC., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Hepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide despite the development of various therapeutic strategies. A better understanding of the mechanisms responsible for HCC initiation and progression is essential for the development of more effective therapies. The cancer stem cell (CSC) model has provided new insights into the development and progression of HCC. CSCs are specialized tumor cells that are capable of self-renewal and have long-term repopulation potential. As they are important mediators of tumor proliferation, invasion, metastasis, therapy resistance, and cancer relapse, the selective targeting of this crucial population of cells has the potential to improve HCC patient outcomes and survival. In recent years, the role of epithelial-to-mesenchymal transition (EMT) in the advancement of HCC has gained increasing attention. This multi-step reprograming process resulting in a phenotype switch from an epithelial to a mesenchymal cellular state has been closely associated with the acquisition of stem cell-like attributes in tumors. Moreover, CSC mediates tumor metastasis by maintaining plasticity to transition between epithelial or mesenchymal states. Therefore, understanding the molecular mechanisms of the reprograming switches that determine the progression through EMT and generation of CSC is essential for developing clinically relevant drug targets. This review provides an overview of the proposed roles of CSC in HCC and discusses recent results supporting the emerging role of EMT in facilitating hepatic CSC plasticity. In particular, we discuss how these important new insights may facilitate rational development of combining CSC- and EMT-targeted therapies in the future.

Background: The border ocellus, or eyespot, is a conspicuous color pattern element in butterfly wings. For two decades, it has been hypothesized that transcription factors such as Distal-less (Dll) are responsible for eyespot pattern development in butterfly wings, based on their expression in the prospective eyespots. In particular, it has been suggested that Dll is a determinant for eyespot size. However, functional evidence for this hypothesis has remained incomplete, due to technical difficulties., Results: Here, we show that ectopically expressed Dll induces ectopic elemental color patterns in the adult wings of the blue pansy butterfly, Junonia orithya (Lepidoptera, Nymphalidae). Using baculovirus-mediated gene transfer, we misexpressed Dll protein fused with green fluorescent protein (GFP) in pupal wings, resulting in ectopic color patterns, but not the formation of intact eyespots. Induced changes included clusters of black and orange scales (a basic feature of eyespot patterns), black and gray scales, and inhibition of cover scale development. In contrast, ectopic expression of GFP alone did not induce any color pattern changes using the same baculovirus-mediated gene transfer system., Conclusions: These results suggest that Dll plays an instructive role in the development of color pattern elements in butterfly wings, although Dll alone may not be sufficient to induce a complete eyespot. This study thus experimentally supports the hypothesis of Dll function in eyespot development.

Background: Merkel cell carcinoma (MCC) is a cutaneous neuroendocrine tumor that may spread via lymphatics and can therefore be staged with sentinel lymph node biopsy (SLNB). MCC is radiosensitive and chemosensitive, although the role of adjuvant therapy is still unclear. We examined the impact of different treatments on the outcome of MCC., Methods: We performed a retrospective review of state cancer registry data from California, Oregon, and Washington of patients diagnosed with primary skin MCC between 1988 and 2012 (n = 4,038). Data were analyzed using Cox regression and Kaplan-Meier methods to examine disease-specific survival., Results: Patients with positive nodes or no documented nodal evaluation had worse survival compared with node-negative patients. No nodal evaluation had decreased survival compared with lymph node evaluation by SLNB. Completion lymph node dissection conferred improved survival in patients with a positive SLNB. In clinically node-negative patients who had a positive SLNB, radiation and chemotherapy did not affect survival., Conclusions: Lymph node evaluation is an important component to MCC treatment. The role of adjuvant radiation and chemotherapy needs further evaluation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo., Results: A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules., Conclusions: Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally examine foreign genes in butterfly wings and also in other non-model insect systems.

Summary: A comparison of research experience, fellowship training, and ultimate practice patterns of general surgery graduates at a university-based surgical residency program. Research experience correlated with pursuing fellowship training and predicted an eventual academic career. More recently, graduates have been able to obtain fellowships without a dedicated research year, perhaps reflecting shifting fellowship training opportunities., Background: We hypothesized that the relationships among dedicated research experience during residency, fellowship training, and career choices is changing as research and fellowship opportunities evolve., Methods: Comparison of research experience, fellowship training, and ultimate practice patterns of general surgery graduates for 2 decades (1990-1999, n = 82; 2000-2009, n = 98) at a university-based residency program. Main outcome measures were number of years and area of research, fellowship training, and practice setting., Results: Compared by decade, graduates became increasingly fellowship-trained (51.2% vs 67.3%; p < 0.05) and pursuit of fellowship training increased for both research and nonresearch participating graduates. The number of residents completing more than 1 year of research doubled (9.8% vs 22.4%, p < 0.05). By decade, the percentage of female graduates increased significantly (22% vs 41%, p = 0.005), with more women participating in dedicated research (17% vs 51%, p < 0.001) and seeking fellowships. The number of graduates going into specialty practice and academic/clinical faculty positions increased over time., Conclusions: Surgical residents have completed more dedicated research years and became increasingly fellowship-trained over time. The proportion of female graduates has increased with similar increases in research time and fellowship training in this subgroup. In the earlier decade, dedicated research experiences during surgical residency correlated with pursuing fellowship training, and predicted an eventual academic career. More recently, graduates have obtained fellowships and academic positions without dedicated research time, perhaps reflecting shifting fellowship opportunities., (Copyright © 2011 Association of Program Directors in Surgery. Published by Elsevier Inc. All rights reserved.)

Urinary tract infection is the commonest bacterial infection in pregnancy. The overall incidence is 5.0-10.0% of all pregnancy. During pregnancy bacterial growth is favoured by increased urinary content of glucose, aminoacids and other nutrients. Other factors responsible for infection are basically related to hormonal effect and mechanical factors. Prolonged stasis of urine in urinary bladder favours growth of micro organism, relaxation of vesico-ureteric junction leads to reflux of urine from bladder to ureter and later up to renal pelvis and later can affect the renal parenchyma affecting the function of kidneys. In addition, some maternal defense mechanism are less effective during pregnancy. Bacteriuria either asymptomatic (5.0%) or symptomatic is common in pregnancy, if left untreated, asymptomatic bacteriuria will lead to acute pyelonephritis in 20.0-30.0%. This may result in abortion, premature delivery, low birth baby and even still birth. About 12.0% of antenatal admission are sepsis due to pyelonephritis. Keeping in mind that UTI in pregnancy leads to increase in maternal morbidity as well as neonatal morbidity and mortality. In this prospective study all asymptomatic consecutive antenatal women were included 200 from each trimester with total of 600 in number to see the incidence in different trimester, most prevalent organisms and it's sensitivity. They were followed up till delivery to see the incidence of asymptomatic bacteriurea in different trimester and its outcome in terms of type of delivery, baby weight, apgar score given at the time of birth and hospital admission for morbidity.

Purpose: Our aim was to determine what specific patient and peri-operative factors contribute to major complications after esophagectomy., Methods: Using the American College of Surgeons National Surgical Quality Improvement Program database, data for esophagectomies between the years 2005 and 2008 were extracted and analyzed. Thirty-day post-operative complications were classified into seven major groups: (1) wound infections, (2) respiratory complications (pneumonia, intubation), (3) cardiac complications, (4) deep venous thrombosis, (5) sepsis/septic shock, (6) re-operation, and (7) death. Univariate analysis and logistic regression modeling were performed to determine if a significant association existed between patient factors or peri-operative factors and these post-operative complications., Results: One thousand thirty-two patients who underwent esophagectomy were identified. Diabetes was the strongest pre-operative independent predictor of death (odds ratio (OR) 10.98; 95% confidence interval (CI) 1.37-1.15, p < 0.1) or respiratory (OR 1.86; 95% CI 1.03-3.29, p = 0.04) or cardiac (OR 5.14; 95% CI 1.93-13.20, p < 0.01) complications following esophagectomy. Thoracotomy performed during the operation was not associated with an increased risk of respiratory or cardiac complications., Conclusions: The major predictors of morbidity after an esophagectomy are the patient factors of diabetes, dyspnea, peripheral vascular disease, and cerebrovascular accident while the peri-operative factors are pre-operative international normalized ratio, contaminated wound classification, and American Society of Anesthesiologists class. Similarly, the major predictors of mortality are diabetes, dyspnea, and age for patient factors and contaminated wound classification for peri-operative factors.

Butterfly wing color patterns can be changed by the application of a temperature shock or pharmacological agents such as tungstate, producing a distinctive type of elemental modification called the TS (temperature shock) type. Heterochronic uncoupling between the signaling and reception steps during the color-pattern determination process has been proposed as a mechanism for TS-type changes. As an extension of this hypothesis, both the parafocal element (PFE) and the eyespot in the same wing compartment are considered to be determined by morphogenic signal(s) emitted from the same eyespot focus. However, these models need to be examined with additional experimental data. Furthermore, there is controversy as to whether the action of tungstate on wing color patterns is direct or Indirect. Using a species of nymphalid butterfly (Junonia orithya), we have devised a simple method for the local application of pharmacological agents directly on developing wings of pupae. Local tungstate application resulted in reduced eyespots and circular dislocated PFEs in the eyespot-less compartments only on the treated wing, demonstrating that tungstate directly induces color-pattern changes on wings. We further examined the eyespot-PFE relationship in normal and cold-shocked Individuals, showing that an eyespot can be superimposed on a PFE and vice versa, probably depending on the timing of their fate determination. Taken together, we propose a two-morphogen model for the normal color-pattern determination, in which the morphogenic signals for the eyespot and PFE are different from each other despite their Identical origin. This two-morphogen model is compatible with the heterochronic uncoupling model for TS-type changes.

Background: Human Immunodeficiency Virus (HIV)/ Acquired Immunodeficiency Syndrome (AIDS) claimed more than 22 million deaths in the past two decades. About 10,000-15,000 Nepalese are expected to die of AIDS related deaths every year in the absence of effective treatment and care. Major cause of mortality and morbidity in HIV infected people is opportunistic infection (OI). Type of pathogen(s) responsible for OI varies from region to region., Objective: This study attempts to find out OI among HIV seropositive patients visiting MTH, Pokhara., Results: Tuberculosis (30%) was found to be most common OI followed by candidiasis (14%). Pulmonary tuberculosis (21.14%) was more common than extra pulmonary tuberculosis (8.92%). Oral candidiasis (8.92%) was more common than oesophageal candidiasis (5.35%)., Conclusion: The study findings indicate that immediate and strong action needs to be taken and guidelines and strategies to tackle the HIV AIDS problems are required and should be equally implemented to achieve a decline in the rate of prevalence of HIV.

Acquisition of full T-cell effector function and memory differentiation requires appropriate costimulatory signals, including ligation of the costimulatory molecule OX40 (TNFRSF4, CD134). Tumors often grow despite the presence of tumor-specific T cells and establish an environment with weak costimulation and immune suppression. Administration of OX40 agonists has been shown to significantly increase the survival of tumor-bearing mice and was dependent on the presence of both CD4 and CD8 T cells during tumor-specific priming. To understand how OX40 agonists work in mice with established tumors, we developed a model to study changes in immune cell populations within the tumor environment. We show here that systemic administration of OX40 agonist antibodies increased the proportion of CD8 T cells at the tumor site in three different tumor models. The function of the CD8 T cells at the tumor site was also increased by administration of OX40 agonist antibody, and we observed an increase in the proportion of antigen-specific CD8 T cells within the tumor. Despite decreases in the proportion of T regulatory cells at the tumor site, T regulatory cell function in the spleen was unaffected by OX40 agonist antibody therapy. Interestingly, administration of OX40 agonist antibody caused significant changes in the tumor stroma, including decreased macrophages, myeloid-derived suppressor cells, and decreased expression of transforming growth factor-beta. Thus, therapies targeting OX40 dramatically changed the tumor environment by enhancing the infiltration and function of CD8 T cells combined with diminished suppressive influences within the tumor.

Background: The expression of OX40 (CD134) on activated CD4+ T cells has been associated with favorable cancer patient outcomes. Because of recent reports that sentinel lymph nodes (SLNs) may represent an immunosuppressive environment, we investigated the expression of OX40 in SLNs from patients with primary cutaneous melanoma., Methods: Samples of peripheral blood lymphocytes and a section of 71 SLNs from 53 patients with clinically node negative melanoma were purified for CD4+ T cells, stained for OX40, and analyzed by flow cytometry., Results: The mean percentage of OX40 on CD4 T cells in the SLNs versus peripheral blood lymphocytes was related indirectly to the T stage of the primary tumor and was decreased in ulcerated primary tumors and positive sentinel nodes., Conclusions: The expression of OX40 on CD4+ T cells in SLNs draining primary melanomas decreased with more advanced tumor features (higher T stage, ulceration) and nodal involvement, suggesting that such tumors may have an immunosuppressive effect on the SLN microenvironment.