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Competing Interests: Conflicts of interest Dr Min is on the advisory boards of Horizon and BMS, and is an investigator for Amgen, BI, BMS, and Priovant. The authors Arora, Kincaid, Sharma, and Mesinkovska have no conflicts of interest to declare.

Competing Interests: Conflicts of interest Dr Min is on the advisory boards of Horizon and BMS. She is an investigator for Amgen, Priovant, and BI. Author Arora and Dr Kesler have no conflicts of interest to declare.

Signal transducers and activators of transcription (STAT) genes are involved in signal mediation of various hormones and cytokines. STAT1 located on chromosome number 2 is involved in mammary gland development and is associated with milk composition traits in bovines. This study aimed to find any relationship and impact of STAT1/ BspHI gene with milk fat and protein yields in a herd of Holstein Friesian (HF) crossbred cattle of sub-tropical climate of Northern India. Milk composition data of 535 adult HF crossbred cows for a period of 12 years was collected from the records maintained at Livestock Farm, Guru Angad Dev Veterinary and Animal Sciences University. First lactation data of 222 animals was chosen for further analysis. After data correction for non-genetic factors (season of calving, period of calving, interaction effect of season and period of calving and age at first calving) these animals were categorised into two groups based on corrected high and low milk fat and protein yields. Forty animals were then selected for blood collection and further laboratory analysis. Amplified using PCR-RFLP technique, the 314 bp STAT1 gene was digested using BspHI restriction enzyme. C-T polymorphism at nucleotide position 201 and 260 of the STAT1 amplicon was observed. At 201, for genotype AA and Aa, the genotypic frequencies were 0.80 and 0.20%. At 260, for genotype BB and Bb, the genotypic frequencies were 0.25 and 0.75%. Least square analysis showed a significant association of all genotypes with milk fat and protein yields. Hence, STAT1 can be used as a potential candidate gene to aid in better animal selection in breeding programmes.

Competing Interests: Conflicts of interest None disclosed.

Competing Interests: Conflicts of interest Dr Min is on the advisory boards of Horizon and BMS and is an investigator for Amgen, Priovant, and BMS. Authors Elghazzawy, Nassir, and Arora have no conflicts of interest to declare.

Background: The COVID-19 pandemic prompted a surge in telehealth utilization. However, language barriers have emerged as a potential obstacle to effective telemedicine engagement, impacting millions of limited English proficient (LEP) individuals. Understanding the role of language spoken in telehealth outcomes is critical, particularly in cancer care, in which consistent follow-up and communication are vital. The primary objective was to assess the impact of telehealth utilization and primary language spoken on clinical outcomes in cancer patients., Methods: This study utilized a retrospective cohort design, encompassing cancer patients seen at the Chao Family Comprehensive Cancer Center between March 1, 2020, and December 31, 2022. The study incorporated both in-person and telehealth visits, examining the association between encounter type and clinical outcomes., Results: The study included 7890 patients with more than one outpatient visit during the study period. There was decreased telehealth utilization in non-English speaking cancer patients throughout the pandemic. Increased telehealth utilization was associated with higher rates of admission, irrespective of cancer type. Additionally, telehealth visits were associated with longer duration of subsequent admissions compared to in-person visits. Spanish-speaking patients utilizing telehealth had higher rates of re-admission compared to English speakers utilizing telehealth. Patients who died had higher rates of telehealth utilization compared to patients who survived., Conclusions and Relevance: This study demonstrates that primary language spoken is associated with differences in telehealth utilization and associated outcomes in cancer patients. These differences suggest that the interplay of telehealth and language could contribute to widening of disparities in clinical outcomes in these populations. The study underscores the need to optimize telehealth usage and minimize its limitations to enhance the quality of cancer care in a telehealth-driven era., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Objective: To assess the effectiveness of an interactive iPad-based educational module (cleft iBook) in enhancing parent/caregiver education related to cleft lip and/or cleft palate (CL/P) care., Design: A prospective study involving pre- and post-intervention surveys., Setting: The study was conducted at a craniofacial clinic in a dedicated children's hospital specifically during initial consultations for CL/P care., Participants: Thirty-two participants (parents/legal guardians/caregivers) ≥18 years of age, English and/or Spanish-speaking, and attending with a child with CL/P., Interventions: Participants were provided with iPads and engaged with the interactive iPad-based educational module. Pre- and post-intervention surveys were administered., Main Outcome Measure(s): The survey assessed the understanding of a CL/P diagnosis and management and usability of the iBook. Survey responses were graded on a 5-point Likert scale. Total scores for pre- and post-intervention surveys were compared., Results: Utilizing the cleft iBook module before consultation significantly enhanced comprehension in multiple domains: prenatal development of CL/P, dento-facial molding, surgical techniques, steps after the initial consultation, postoperative care, and the necessity of long-term care for affected children ( P  < .01). Cumulative survey scores increased by 10.2 points, reflecting significantly improved responses regarding overall comprehension ( P  < .001)., Conclusions: The interactive, iPad-based cleft educational module emerges as a viable, digital strategy for providing education and empowerment to parents and caregivers navigating the challenges of caring for a child with CL/P. The cleft iBook serves as a readily accessible resource, fostering connections among the child, caregivers, and care team., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Immediate lymphatic reconstruction (ILR) has traditionally required a fluorescent-capable microscope to identify lymphatic channels used to create a lymphaticovenous bypass (LVB). Herein, a new alternative method is described, identifying lymphatic channels using a commercially available handheld fluorescence imaging device., Methods: This was a single-center study of consecutive patients who underwent ILR over a 1-year period at a tertiary medical center. Intradermal injection of fluorescent indocyanine green dye was performed intraoperatively after axillary or inguinal lymphadenectomy. A handheld fluorescent imaging device (SPY-PHI, Stryker) rather than a fluorescent-capable microscope was used to identify transected lymphatic channels. Data regarding preoperative, intraoperative, and outcome variables were collected and analyzed., Results: The handheld fluorescent imaging device was successfully able to identify transected lymphatic channels in all cases (n = 15). A nonfluorescent-capable microscope was used to construct the LVB in 14 cases. Loupes were used in one case. In 13 cases, ILR was unilateral. In two cases, bilateral ILR was performed in the lower extremities. All upper extremity cases were secondary to breast cancer (n = 7). Lower extremity cases (n = 8) included extramammary Paget disease of the penis, ovarian cancer, vulvar squamous cell carcinoma, squamous cell carcinoma of unknown origin, soft tissue sarcomas, cutaneous melanoma, and porocarcinoma., Conclusions: ILR, using indocyanine green injection with a handheld fluorescent imaging device, is both safe and effective. This method for intraoperative identification of lymphatic channels was successful, and LVB creation was completed in all cases. This approach makes ILR feasible when a fluorescent-capable microscope is unavailable, broadening access to more patients., Competing Interests: The authors have no financial interest to declare in relation to the content of this article. Disclosure statements are at the end of this article, following the correspondence information., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Background: Current literature has sparse recommendations that guide social networking practices in plastic surgery. To address this, we used natural language processing and sentiment analysis to investigate the differences in plastic surgery-related terms and hashtags on Twitter., Methods: Over 1 million tweets containing keywords #plasticsurgery, #cosmeticsurgery, and their non-hashtagged versions plastic surgery and cosmetic surgery were collected from the Twitter Gardenhose feed spanning from 2012 to 2016. We extracted the average happiness/positivity (h-avg) using hedonometrics and created word-shift graphs to determine influential words., Results: The most popular keywords were plastic and cosmetic surgery, comprising more than 90% of the sample. The positivity scores for plastic surgery, cosmetic surgery, #plasticsurgery, and #cosmeticsurgery were 5.72, 6.00, 6.17, and 6.18, respectively. Compared to plastic surgery, the term cosmetic surgery was more positive because it lacked antagonistic words, such as "fake," "ugly," "bad," "fails," and "wrong." For similar reasons, #plasticsurgery and #cosmeticsurgery were more positively associated than their non-hashtagged counterparts., Conclusion: Plastic surgery-related hashtags are more positively associated than their non-hashtagged versions. The language associated with such hashtags suggests a different user profile than the public and, given their underutilization, remain viable channels for professionals to achieve their diverse social media goals., Level of Evidence V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 ., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature and International Society of Aesthetic Plastic Surgery.)

Background: Sentinel node biopsy is performed to stage clinically and radiographically occult regional disease in cases of malignancy, including cutaneous cancer such as melanoma. The use of tumescent anesthesia with sentinel node biopsy for cutaneous malignancies has not been well studied. Therefore, we aimed to compare the rate of successful sentinel node identification and estimated blood loss between patients who underwent sentinel node biopsy with and without the use of tumescent anesthesia., Methods: A retrospective review was conducted of a prospectively maintained single-institution database of all patients who underwent reconstruction after the extirpation of a cutaneous malignancy over an 18-month period. Patient demographics, tumor histology, and characteristics, indication for and success of sentinel node biopsy, use of tumescent anesthesia, and total estimated blood loss were examined., Results: Sentinel node biopsy was performed in 15 of 39 patients (38.5%) receiving tumescent anesthesia compared with 6 of 26 patients (23.1%) not receiving it (p = 0.19). Sentinel node biopsy had a success rate of 100% in the tumescent and nontumescent anesthesia groups. The mean estimated blood loss in the tumescent anesthesia group was 36.7 mL versus 59.6 mL in the nontumescent anesthesia group (p < 0.001). Complication rates were comparable between the tumescent anesthesia (12.8%) and nontumescent anesthesia (19.2%) groups (p = 0.48)., Conclusion: The use of tumescent anesthesia in cutaneous malignancy extirpation and immediate reconstruction was not associated with a decreased sentinel node identification rate or change in complication rates. However, tumescent anesthesia was associated with a decrease in the estimated blood loss., Competing Interests: Declaration of Competing Interest All authors do not have any financial or personal relationships to disclose., (Copyright © 2023 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Background: Graduate medical education during the COVID-19 pandemic has seen the shift to a "virtual learning" format in many aspects of training. The purpose of this study was to describe the perceived strengths and weaknesses of virtual learning compared with a conventional, in-person format., Methods: A 45-question survey was sent to independent and integrated plastic surgery residents and postresidency fellows nationally. The survey collected basic demographic information and evaluated three general categories of virtual learning in comparison to an in-person format: (1) time, (2) learning proficiency, and (3) collaboration., Results: In total, 108 surveys were submitted from 48 different training programs. Participants reported that virtual learning was more efficient (mean: 3.9), conducive to more free time (mean: 3.9), and a more comfortable medium for expressing opinions (mean: 3.5) and asking questions (mean: 3.6) compared with an in-person format. When stratified between training levels, the PGY 1-3 group reported more difficulties in exam preparedness ( P = 0.05), motivation to study ( P = 0.01) and less time-saving benefits ( P = 0.05) with a virtual format than the PGY 4+ group. Lastly, respondents who had higher self-reported levels of multitasking were found to have lower mean Likert scale scores on all questions related to "time," "learning proficiency," and "collaboration" ( P < 0.01)., Conclusions: A virtual and in-person hybrid approach toward plastic surgery education may be beneficial for encouraging flexibility. Our results demonstrate impairment with collaboration and learning proficiency with a virtual format, especially with increased multitasking, but increased comfort with expressing opinions and asking questions., Competing Interests: The authors have no financial interest to declare in relation to the content of this article. The authors have no relationships to disclose. No funding was provided for this article., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Background: The purpose of this study was to evaluate outcomes and complications associated with infant ear molding at a single institution., Methods: We conducted a retrospective chart review of all infants who underwent ear molding using the EarWell Infant Ear Correction System with pediatric plastic surgery from October 2010 to March 2021. Types of ear anomalies, age at initiation, duration of treatment, gaps in treatment, comorbidities, and complications were extracted for included patients. The primary outcomes assessed were degree of ear anomaly correction and incidence of skin complications. Parents were also sent a questionnaire regarding their long-term satisfaction with the ear molding treatment process., Results: A total of 184 ears of 114 patients meeting inclusion criteria were treated during the study period. Mean age at treatment initiation was 21 days, and average duration of treatment was 40 days. Helical rim deformities (N = 50 ears) and lop ear (N = 40 ears) were the most common anomalies. A total of 181 ears (98.4%) achieved either a complete (N = 125 ears, 67.9%) or partial correction (N = 56 ears, 30.4%). The most common complications were eczematous dermatitis (N = 27 occurrences among 25 ears, 13.6%) and pressure ulcers (N = 23 occurrences among 21 ears, 12.5%). Infants who experienced a complication were 3.36 times more likely to achieve partial relative to complete correction ( P < 0.001; 95% confidence interval 1.66-6.81)., Conclusion: Ear molding is an effective treatment strategy for infant ear anomalies, with most patients achieving complete correction., Competing Interests: The authors have no financial interest to declare in relation to the content of this article., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Approximately 20% of retained foreign bodies are surgical needles. Retained macro-needles may become symptomatic, but the effect of microsurgical needles is uncertain. We present the first animal model to simulate microsurgical needle retention. Given a lack of reported adverse outcomes associated with macro-needles and a smaller cutting area of microsurgical needles, we hypothesized that microsurgical needles in rats would not cause changes in health or neurovascular compromise., Methods: Male Sprague-Dawley rats (x̄ weight: 288.9 g) were implanted with a single, 9.0 needle (n = 8) or 8.0 needle (n = 8) orthogonal to the right femoral vessels and sutured in place. A control group (n = 8) underwent sham surgery. Weekly, a cumulative health score evaluating body weight, body condition score, physical appearance, and behavior for each rat was determined. Infrared thermography (°C, FLIR one) of each hindlimb and the difference was obtained on postoperative days 15, 30, 60, and 90. On day 90, animals were euthanatized, hindlimbs were imaged via fluoroscopy, and needles were explanted., Results: The mean, cumulative health score for all cohorts at each weekly timepoint was 0. The mean temperature difference was not significantly different on postoperative days 15 ( P = 0.54), 30 ( P = 0.97), 60 ( P = 0.29), or 90 ( P = 0.09). In seven of eight rats, 8.0 needles were recovered and visualized on fluoroscopy. In six of eight rats, 9.0 needles were recovered, but 0/8 needles were visualized on fluoroscopy., Conclusions: Microsurgical needle retention near neurovascular structures may be benign, and imaging for needles smaller than 8.0 may be futile. Further studies should explore microsurgical needle retention potentially through larger animal models., Competing Interests: The authors have no financial interest to declare in relation to the content of this article. Disclosure statements are at the end of this article, following the correspondence information., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Accurate quantification based on nucleic acid amplification is necessary to avoid the spread of pathogens, making early diagnosis essential. Droplet digital PCR (ddPCR) stands out for absolute parasite quantification because it combines microfluidics with the TaqMan test. This helps deliver maximum accuracy without needing a reference curve. This study assessed the efficacy of ddPCR as a detection tool for the bovine theileriosis (BT) caused by Theileria parasites. We developed and validated a duplex ddPCR method that detects and quantifies the Theileria genus (18S rRNA) and identifies clinically significant Theileria annulata parasites (TaSP) in experimental and clinical samples. ddPCR was shown to be as effective as qPCR throughout a 10-fold sample dilution range. However, ddPCR was more sensitive than qPCR at lower parasite DNA concentrations and reliably assessed up to 8.5 copies/µL of the TaSP gene in the infected DNA (0.01 ng) samples. The ddPCR was very accurate and reproducible, and it could follow therapeutic success in clinical cases of theileriosis. In conclusion, our ddPCR assays were highly sensitive and precise, providing a valuable resource for the study of absolute parasite quantification, drug treatment monitoring, epidemiological research, large-scale screening, and the identification of asymptomatic parasite reservoirs in the pursuit of BT eradication.

The domestic dog has been given considerable attention as a system for investigating the genetics of human diseases. Population diversity and breed structure are unique features that make dogs particularly amenable to genetic studies. Dogs show distinguished features of breed-specific homogeneity, which is associated with striking interbreed heterogeneity. This review discusses the significance of studying the genetic maps, genome-wide association studies (GWAS), and usefulness of this species as an animal model. Most canine genetic disorders are similar to those of humans, including inherited, psychiatric, and genetic disorders. In addition to revealing new candidate genes, canine models allow access to experimental resources, such as cells, tissues, and even live animals, for research and intervention purposes., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Humins are carbonaceous, polymeric byproducts formed during the acid-catalyzed condensed phase transformation of biomass-derived moieties and are responsible for significant carbon loss and catalyst deactivation. There exists very limited knowledge about their formation chemistry and composition. Infrared spectra of humins formed during the dehydration of glucose/fructose to 5-HMF show that the furan ring and the hydroxy methyl group of 5-HMF are present in humins, but the carbonyl group is not. Based on this, aldol addition and condensation between 5-HMF and other derived species are proposed as the main reactions that initiate humin formation. Hence, in this work, density functional theory (DFT)-based calculations are performed to compute the reaction pathways, activation barriers, and reaction free energies associated with all elementary reaction steps in the 5HMF-initiated, acid-catalyzed reactions leading to humin formation. The humin formation is initiated with the rehydration of HMF to form 2,5-dioxo-6-hydroxy-hexanal or DHH (key promoter of humin formation), followed by its keto-enol tautomerization and aldol addition and condensation with HMF. The rate-determining step in this pathway is the aldol-addition reaction between the DHH-derived enols with 5-HMF. Within the implicit solvation approximation, the formation of the 5-HMF-DHH dimer is slightly endergonic, whereas the 5-HMF rehydration leading to DHH is thermodynamically downhill. This mechanistic understanding of initiation reactions for humins could pave the way to screen and design solvent and catalyst systems to deter their formation., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Anaplasmosis, an infectious vector borne rickettsial disease caused by different species of Anaplasma transmitted through mechanical and biological (tick) processes has a great concern to livestock industry due to its associated economic losses. The current cross sectional comprehensive epidemiological study was conducted from August 2020 to November 2021 on 391 (277 cattle and 114 buffaloes) bovines from different districts of five agro climatic zones of Punjab state. Classical microscopic examination of Romanowsky stained blood smears showed an occurrence of 13.00%, while DNA amplification targeting major surface protein (msp5) of Anaplasma marginale revealed the 382 bp amplicon in 32.48% samples. Zone based molecular prevalence of A. marginale was highest in the Undulating zone (41.93%) and least in the Sub-Mountain zone (18.84%). The prevalence in Central plain zone, Western plain zone, and Western zone were 40.15, 30.95 and 29.91%, respectively. An overall molecular prevalence of A. marginale was 34.52, 32.75 and 20.0% in young, adults and calves, respectively, the difference being non-significant. Anaplsma marginale was more prevalent in unorganized farms (38.22%; 60/157) than organized farms (28.63%; 66/234). Risk factors analysis revealed young, female cattle at unorganized farms were more prone to anaplasmosis. Out of 127 positive samples, four samples were customed to sequencing revealed 98-99% homology with published sequences for other available global isolates. Multi-single nucleotide polymorphisms (SNP) were observed in the sequence of two samples when aligned with the reference sequence from the NCBI database (CP023731)., (Copyright © 2022. Published by Elsevier B.V.)

The techniques used to make preoperative markings before soft tissue reconstruction have remained relatively unchanged since the earliest years of plastic surgery. Using skin-marking pens, many surgeons continue to draw markings freehand as "best estimates" before their first incisions. Although efficient for the experienced surgeon, this strategy may prove challenging for residents and trainees striving to learn and maintain consistency while replicating the intricate geometries of flap markings. To address this need, Operating Room Stencil was developed as a novel tool for digitally planning flap markings that may then be projected onto contoured surfaces such as the human body. As a cost-free mobile application, Operating Room Stencil is widely accessible to the medical community and offers educational captions for a majority of the flaps featured in its database. Users can plot relaxed skin tension lines onto uploaded facial images, thus enabling surgeons to orient surgical markings in a way that optimizes scar formation and reduces wound contraction. Although originally intended to appeal to trainees as a reliable way to learn about flaps and practice their technique, Operating Room Stencil may prove useful even among more experienced surgeons striving to further perfect their visualization and execution of flap markings., Competing Interests: Disclosure: The authors have no financial interest to declare in relation to the content of this article. The development of OR-Stencil was fully funded by a grant from the Aesthetic Surgery Education and Research Foundation entitled “New Innovation for Projected Flap Design.” The foundation had no involvement in project development., (Copyright © 2021 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Hydroxyurea (HU) and thalidomide have been reported to improve clinical and hematological parameters in transfusion-dependent beta thalassemia (TDT). Therefore, we retrospectively analyzed the combination of HU and thalidomide in 140 transplant ineligible TDT, ≥ 10 years old, visiting our thalassemia clinic between October 2014 and November 2019. Responses were defined as maintenance of hemoglobin ≥9gm/dl without transfusion as complete response (CR) and with at least 50% reduction in transfusion burden as partial response (PR). Patients with less than 50% transfusion burden reduction for consecutive 6 months of therapy were defined as non-responders (NR), and treatment was discontinued thereafter. Primary end point was overall response rate (ORR) at last follow-up. At median follow-up of 22.6 (95% CI 16.4-28.7) months, 76 (57.2%) patients achieved CR and 19 (14.3%) achieved PR, accounting to an ORR of 71.5%. Among responders at last follow-up, a significant increase in the post-treatment hemoglobin (0.88±0.37gm/dl, p<0.0001) and drop in serum ferritin (-1490.5ng/ml, p<0.0001) were observed. Median time to CR was 124 (95% CI 75.3-172.6) days. Median longest continuous CR was 791 (95% CI 662.2-919.7) days. Common toxicities observed were sedation (25%), hyperbilirubinemia {(23.57%, grade 3/4 =17 (12.14%)}, and constipation (22.8%). Nearly three-fourth of the patients has responded with majority having CR. Adverse events are a concern; hence, regular close monitoring is a prerequisite.

Background: Apicomplexan parasite Theileria annulata causes significant economic loss to the livestock industry in India and other tropical countries. In India, parasite control is mainly dependent on the live attenuated schizont vaccine and the drug buparvaquone. For effective disease control, it is essential to study the population structure and genetic diversity of the Theileria annulata field isolates and vaccine currently used in India. Methodology/Results: A total of 125 T. annulata isolates were genotyped using 10 microsatellite markers from four states belonging to different geographical locations of India. Limited genetic diversity was observed in the vaccine isolates when compared to the parasites in the field; a level of geographical substructuring was evident in India. The number of genotypes observed per infection was highest in India when compared to other endemic countries, suggesting high transmission intensity and abundance of ticks in the country. A reduced panel of four markers can be used for future studies in these for surveillance of the T. annulata parasites in India. Conclusion: High genetic variation between the parasite populations in the country suggests their successful spread in the field and could hamper the disease control programs. Our findings provide the baseline data for the diversity and population structure of T. annulata parasites from India. The low diversity in the vaccine advocates improving the current vaccine, possibly by increasing its heterozygosity. The reduced panel of the markers identified in this study will be helpful in monitoring parasite and its reintroduction after Theileria eradication., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Roy, Bhandari, Barman, Kumar, Bhanot, Arora, Singh and Sharma.)

Hippocampal spiking sequences encode external stimuli and spatiotemporal intervals, linking sequential experiences in memory, but the dynamics controlling the emergence and stability of such diverse representations remain unclear. Using two-photon calcium imaging in CA1 while mice performed an olfactory working-memory task, we recorded stimulus-specific sequences of "odor-cells" encoding olfactory stimuli followed by "time-cells" encoding time points in the ensuing delay. Odor-cells were reliably activated and retained stable fields during changes in trial structure and across days. Time-cells exhibited sparse and dynamic fields that remapped in both cases. During task training, but not in untrained task exposure, time-cell ensembles increased in size, whereas odor-cell numbers remained stable. Over days, sequences drifted to new populations with cell activity progressively converging to a field and then diverging from it. Therefore, CA1 employs distinct regimes to encode external cues versus their variable temporal relationships, which may be necessary to construct maps of sequential experiences., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Lysyl oxidase (LOX) is an extracellular metalloenzyme which mediates crosslinking of collagen and elastin. It has been reported to play a pivotal role in cancer metastasis especially in women suffering from breast cancer. The present study is the first to evaluate the gene expression levels of LOX by Real time-polymerase chain reaction (Real time-PCR) in dogs with mammary tumor besides molecular cloning and expression of canine lysyl oxidase gene (lox). Real time-PCR studies showed a significant upregulation (threefold higher) of lox in mammary tumor cases as compared to healthy dogs indicating its possible diagnostic and prognostic role in canine mammary tumors (CMTs). Cloning and sequencing of lox gene revealed 1230 bp CDS which is mostly conserved in C-terminal region. Sequence analysis of canine lox showed that it shares 99% homology with the predicted sequence available on NCBI and had greatest identity with the lox gene from cat. Protein structure predicted with homology modelling was validated by Ramachandran plot analysis which revealed most (approximately 95%) of the amino acids in favoured region. Additionally, recombinant lysyl oxidase expressed as His-tagged fusion protein in prokaryotic expression vector (pPROExHTa) was used in an ELISA for detection of circulating protein LOX in serum of CMT subjects. Receiver operating characteristics analysis of the ELISA revealed high sensitivity (90%) and specificity (85%) with histopathology as reference standard. Taken together, we propose LOX as a diagnostic biomarker and a putative prognostic candidate in CMT cases.

The central dogma of gene expression propounds that DNA is transcribed to mRNA and finally gets translated into protein. Only 2-3% of the genomic DNA is transcribed to protein-coding mRNA. Interestingly, only a further minuscule part of genomic DNA encodes for long non-coding RNAs (lncRNAs) which are characteristically more than 200 nucleotides long and can be transcribed from both protein-coding (e.g. H19 and TUG1 ) as well as non-coding DNA by RNA polymerase II. The lncRNAs do not have open reading frames (with some exceptions), 3`-untranslated regions (3'-UTRs) and necessarily these RNAs lack any translation-termination regions, however, these can be spliced, capped and polyadenylated as mRNA molecules. The flexibility of lncRNAs confers them specific 3D-conformations that eventually enable the lncRNAs to interact with proteins, DNA or other RNA molecules via base pairing or by forming networks. The lncRNAs play a major role in gene regulation, cell differentiation, cancer cell invasion and metastasis and chromatin remodeling. Deregulation of lncRNA is also responsible for numerous diseases in mammals. Various studies have revealed their significance as biomarkers for prognosis and diagnosis of cancer. The aim of this review is to overview the salient features, evolution, biogenesis and biological importance of these molecules in the mammalian system., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Theileria annulata is an intracellular parasite that causes active and latent forms of bovine theileriosis. Diagnosis of the disease is primarily based on traditional methods such as microscopy, however, PCR based methods have proven to be superior in the absence of clear disease symptoms. However, diagnosis is difficult in cases of lower parasitaemia by conventional PCR. Hence, a rapid and sensitive method which can detect early infection and low parasite load is required. Therefore, we have developed an absolute quantification based real-time PCR (qPCR) assay. Reference standard curve using recombinant plasmids of a host (hprt) and a parasite gene (tasp) was constructed, and the assay was initially standardised using in vitro T. annulata cell lines. Further, 414 blood samples from suspected theileriosis cases were also evaluated using qPCR. The assay can estimate host to parasite ratios, calculate parasitaemia and treatment effectiveness in the clinical cases of theileriosis. In comparison with the conventional PCR results, 44 additional positive cases were found. Therefore, the assay holds importance in a clinical setting due to its ability to quantify the parasite load in clinical samples. It may be further used in distinguishing active and latent theileriosis infections and detection of drug resistance in the field.

Fast pyrolysis is a promising technology for the production of renewable fuels and chemicals from lignocellulosic biomass. The product distribution (bio-oil, char) and the composition of bio-oil are significantly influenced by the presence of naturally occurring alkali and alkaline-earth metals (AAEMs). In this paper, we investigate, at the molecular level, the influence of Na(I), K(I), Ca(II), and Mg(II) ions on glycosidic bond breaking reactions using density functional theory. Glycosidic bond breaking reactions are categorized as direct C-O breaking mechanisms, namely, transglycosylation, glycosylation, and ring contraction and the two-step pathways, which include the mannose pathway, dehydration, and ring opening. Our calculations show that in the absence of metal, transglycosylation and dehydration pathways (activation barriers ∼55 kcal.mol -1 ) are kinetically most facile. The linkage type (α- or β-1,4) has an insignificant effect on kinetics of glycosidic bond cleavage. Mg(II) ions have a pronounced effect on lowering the activation barriers of glycosylation, ring contraction, and the mannose pathway, requiring activation enthalpies of 32-52 kcal.mol -1 . Conversely, Mg(II) and Ca(II) ions inhibit the dehydration pathway. Na(I) and K(I) ions do not significantly influence the activation barriers of glycosidic bond cleavage reactions, as the reduction is only about 5-10 kcal.mol -1 . Thus, AAEM ions exhibit different catalytic effects on glycosidic bond breaking reactions.

An enantioselective organocatalytic multicomponent reaction of aldehydes, ketones, and Meldrum's acid has been developed. A cinchona-based primary amine (1 mol %) catalyses the multicomponent reaction via the formation of the Knoevenagel product and a chiral enamine to form enantiopure δ-keto Meldrum's acids in a tandem catalytic pathway. An adverse effect of higher catalyst loading and longer reaction time on enantioselectivity was studied. This mild protocol provides an easy access to enantiopure carboxylic acids, esters and amides and the method is scalable on a gram quantity. DFT calculations were carried out on the proposed reaction mechanism and they were in close agreement with the experimental results., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Aim: Newcastle disease (ND) is considered one of the most important poultry diseases with chicken morbidity and mortality rates up to 100%. Current vaccination programs allow the use of live attenuated vaccines in the field to protect against the disease, which alone is inefficient and requires repeat booster doses. Toll-like receptor agonists (e.g., lipopolysaccharide [LPS]) as adjuvants are the ones, most extensively studied and have shown to be very promising in delivering a robust balanced immune response. In the present study, we have evaluated the potential of LPS to elicit a strong immune response with respect to the elicitation of both Th1 (cell-mediated) and Th2 (humoral) immune arms., Materials and Methods: A total of 72 apparently healthy 1-day-old indigenous unvaccinated chicks were randomly divided into six experimental Groups A to F (n=12). At 8-week of age chicks in Group A, C, and E were vaccinated with live attenuated La Sota strain ND vaccine along with LPS, bovine serum albumin, and normal saline solution, respectively, and those in Group B, D, and E were kept separately without vaccination. Sampling was done on days 0, 1, 3, 7, 14, 21, 35, and 60 after vaccination. After vaccination and respective adjuvant application, Th1 and Th2 cytokine expression were measured in mRNA of both blood and tissue samples., Results: The results were validated by, hemagglutination inhibition and enzyme-linked immunosorbent assay tests, to check for the humoral as well as cell-mediated immune response in blood serum levels. The results showed an increase in mRNA expression of the Th1 biased cytokines in Group A (LPS+NDV) as compared to the control groups. Similar mRNA expression pattern was seen in blood as well as tissue samples. Validation of results also indicates an increase in Cell-mediated Immunity as well as a humoral immune response in Group A (LPS+NDV)., Conclusion: The results of the study provided enough evidence to consider LPS as a potential vaccine adjuvants candidate against ND in chicken.

MicroRNAs (miRNAs) are small (19-25 base long), non-coding RNAs that regulate post-transcriptional gene expression by cleaving targeted mRNAs in several eukaryotes. The miRNAs play vital roles in multiple biological and metabolic processes, including developmental timing, signal transduction, cell maintenance and differentiation, diseases and cancers. Experimental identification of microRNAs is expensive and lab-intensive. Alternatively, computational approaches for predicting putative miRNAs from genomic or exomic sequences rely on features of miRNAs viz. secondary structures, sequence conservation, minimum free energy index (MFEI) etc. To date, not a single miRNA has been identified in bubaline (Bubalus bubalis), which is an economically important livestock. The present study aims at predicting the putative miRNAs of buffalo using comparative computational approach from buffalo whole genome shotgun sequencing data (INSDC: AWWX00000000.1). The sequences were blasted against the known mammalian miRNA. The obtained miRNAs were then passed through a series of filtration criteria to obtain the set of predicted (putative and novel) bubaline miRNA. Eight miRNAs were selected based on lowest E-value and validated by real time PCR (SYBR green chemistry) using RNU6 as endogenous control. The results from different trails of real time PCR shows that out of selected 8 miRNAs, only 2 (hsa-miR-1277-5p; bta-miR-2285b) are not expressed in bubaline PBMCs. The potential target genes based on their sequence complementarities were then predicted using miRanda. This work is the first report on prediction of bubaline miRNA from whole genome sequencing data followed by experimental validation. The finding could pave the way to future studies in economically important traits in buffalo., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: The present study investigated the immunogenic potential of different cationic liposome formulations with a DNA plasmid encoding Pfs25, a malaria transmission-blocking vaccine candidate., Methods: Pfs25 plasmid DNA was complexed with cationic liposomes to produce lipoplexes at different charge ratios of the cationic lipid head group to the nucleotide phosphate (N:P). The formation of lipoplexes was visualized by Cryogenic-TEM. Confocal microscopy of lipoplexes formed with GFP encoding plasmid DNA, and flow cytometry was used to determine their in vitro transfection capability. Two different lipoplex formulations using plasmid DNA encoding Pfs25 were evaluated for in vivo immunogenicity after intramuscular administration in Balb/c mice. Immune sera were analyzed by ELISA., Results: The results demonstrated that the cationic liposome-mediated DNA immunization with an N:P charge ratio of 1:3 (anionic lipoplexes) is more effective than the use of naked plasmid DNA alone. No antibody response was observed when lipoplexes with a higher N:P charge ratio of 10:3 (cationic lipoplexes) were used. Trehalose was added to some lipoplex formulations as a cryoprotectant and adjuvant, but it did not yield any further improvement of immunogenicity in vivo., Conclusions: The results suggest that Pfs25 plasmid DNA delivered as lipoplexes at a charge ratio of 1:3 elicited strong immunogenicity in mice and may be improved further to match the immune responses of DNA vaccines administered by in vivo electroporation.

Heat stress is an important domain of research in livestock due to its negative impact on production and disease resistance. The augmentation of stress in the body stimulates the antioxidative activity comprising various enzymes (viz., catalase, superoxide dismutase), metabolites (reduced glutathione, etc.), vitamins, minerals, etc. to combat the situation. The major key players involved in regulation of heat shock response in eukaryotes are the transcription factors, called as heat shock factors (HSF). They activate the heat shock protein (HSP) genes by binding to their promoters. Lymphocytes are considered to be the best model to evaluate the immunity in any living body as it contains plethora of white blood cells (WBCs).In this study, the peripheral blood mononuclear cells (PBMC) obtained from non-lactating Sahiwal vis-à-vis crossbred (Holstein Friesian × Sahiwal) cattle with 75% or more exotic inheritance were subjected to heat shock at 39, 41, and 43 °C in three different incubators, in vitro. The cell count and viability test of pre and post heat stress of concerned PBMCs indicated that the crossbreeds are more prone to heat stress as compared to Sahiwal. The reverse transcription PCR (qRT-PCR) expression data revealed an increment in HSF1 expression at 41 °C which subsequently declined (non-significantly) at 43 °C in both breeds post 1 h heat shock. However, the association between the HSF 1 expression and antioxidative activity through correlation analysis was found to be non-significant (P < 0.05), though enzymatic activity appeared to behave in a similar fashion in both breeds at 5% level of significance (P < 0.05). This rule out the role of HSF1 expression level on the activity of enzymes involved in oxidative stress in vitro in zebu and crossbred cattle.

This study reports, for the first time, development of tyrosine kinase inhibitor-loaded, thermosensitive liposomes (TKI/TSLs) and their efficacy for treatment of renal cell carcinoma when triggered by focused ultrasound (FUS). Uptake of these nanoparticles into renal cancer cells was visualized with confocal and fluorescent imaging of rhodamine B-loaded liposomes. The combination of TKI/TSLs and FUS was tested in an in vitro tumor model of renal cell carcinoma. According to MTT cytotoxic assay and flow cytometric analysis, the combined treatment led to the least viability (23.4% ± 2.49%, p < 0.001), significantly lower than that observed from treatment with FUS (97.6% ± 0.67%, not significant) or TKI/TSL (71.0% ± 3.65%, p < 0.001) at 96 h compared to control. The importance of this unique, synergistic combination was demonstrated in viability experiments with non-thermosensitive liposomes (TKI/NTSL + FUS: 58.8% ± 1.5% vs. TKI/TSL + FUS: 36.2% ± 1.4%, p < 0.001) and heated water immersion (TKI/TSL + WB43°: 59.3% ± 2.91% vs. TKI/TSL + FUS: 36.4% ± 1.55%, p < 0.001). Our findings coupled with the existing use of FUS in clinical practice make the proposed combination of targeted chemotherapy, nanotechnology, and FUS a promising platform for enhanced drug delivery and cancer treatment., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The surface chemistry and topography govern the spreading of liquids on a solid. When an oil drop makes a contact angle, θ > 90° on a solid surface, the solid is termed as oleophobic. Adding roughness to an inherently oleophobic surface enhances its oil dewetting and can lead to superoleophobicity when θ > 150°. In this study, we introduce the concept of a two-tier hierarchical roughness on the surface of soft materials such as hydrogels by forming the patterned inverse replica of breath figure polymer films impregnated with nanoparticles. The directed deposition of nanoparticles in the breath figure pores is accomplished by an aerosol assisted technique that exclusively leads to deposition within the pores and filling of the pores. The inverse replica of such impregnated films exhibits a close packed hexagonally structured second tier of surface roughness which directly leads to a superoleophobic surface. Since these structures have well defined geometries, it is possible to estimate the contact angle by assuming a partial wetting of the oil drop in a 'fakir' state on the rough surface. The estimation is in good agreement with the experimental contact angle value. While the work demonstrates a facile method to impart superoleophobicity to a hydrogel surface, it also demonstrates new methods to imbue breath figure pores with functional materials that can be easily transferred to the pores of the inverse replica.

Aim: The active domains (TIR and NACHT) of the pattern recognition receptors (PRRs: Toll-like receptors [TLRs] and nucleotide-binding oligomerization domain [NOD]-like receptors [NLR], respectively) are the major hotspots of evolution as natural selection has crafted their final structure by substitution of residues over time. This paper addresses the evolutionary perspectives of the TLR and NLR genes with respect to the active domains in terms of their chronological fruition, functional diversification, and species-specific stipulation., Materials and Methods: A total of 48 full-length cds (and corresponding peptide) of the domains were selected as representatives of each type of PRRs, belonging to divergent animal species, for the biocomputational analyses. The secondary and tertiary structure of the taurine TIR and NACHT domains was predicted to compare the relatedness among the domains under study., Results: Multiple sequence alignment and phylogenetic tree results indicated that these host-specific PRRs formed entirely different clusters, with active domains of NLRs (NACHT) evolved earlier as compared to the active domains of TLRs (TIR). Each type of TLR or NLR shows comparatively less variation among the animal species due to the specificity of action against the type of microbes., Conclusion: It can be concluded from the study that there has been no positive selection acting on the domains associated with disease resistance which is a fitness trait indicating the extent of purifying pressure on the domains. Gene duplication could be a possible reason of genesis of similar kinds of TLRs (virus or bacteria specific).

High-intensity focused ultrasound (HIFU) can locally ablate biological tissues such as tumors, i.e., induce their rapid heating and coagulative necrosis without causing damage to surrounding healthy structures. It is widely used in clinical practice for minimally invasive treatment of prostate cancer. Nonablative, low-power HIFU was established as a promising tool for triggering the release of chemotherapeutic drugs from temperature-sensitive liposomes (TSLs). In this study, we combine ablative HIFU and thermally triggered chemotherapy to address the lack of safe and effective treatment options for elderly patients with high-risk localized prostate cancer. DU145 prostate cancer cells were exposed to chemotherapy (free and liposomal Sorafenib) and ablative HIFU, alone or in combination. Prior to cell viability assessment by trypan blue exclusion and flow cytometry, the uptake of TSLs by DU145 cells was verified by confocal microscopy and cryogenic scanning electron microscopy (cryo-SEM). The combination of TSLs encapsulating 10 μM Sorafenib and 8.7W HIFU resulted in a viability of less than 10% at 72 h post-treatment, which was significant less than the viability of the cells treated with free Sorafenib (76%), Sorafenib-loaded TSLs (63%), or HIFU alone (44%). This synergy was not observed on cells treated with Sorafenib-loaded nontemperature sensitive liposomes and HIFU. According to cryo-SEM analysis, cells exposed to ablative HIFU exhibited significant mechanical disruption. Water bath immersion experiments also showed an important role of mechanical effects in the synergistic enhancement of TSL-mediated chemotherapy by ablative HIFU. This combination therapy can be an effective strategy for treatment of geriatric prostate cancer patients.

Indoxacarb is commonly used to effectively control pests, cockroaches, termites, fleas, and houseflies. Although the toxicological profile of indoxacarb had already been well characterized, we examined the possible toxicological interaction with indoxacarb and endotoxin. Male Swiss albino mice aged 8-10 weeks were orally administered indoxacarb dissolved in groundnut oil at 4 mg/kg/day and 2 mg/kg/day for 90 days. On day 91, five animals from each group were challenged with lipopolysaccharides (LPS) at 80 µg/mouse, administered intranasally. Indoxacarb at 4 mg/kg significantly decreased Total leukocyte count, lymphocytopenia, and neutrophilia. Both doses of indoxacarb combined with LPS resulted in significant lymphocytopenia. Indoxacarb did not produce DNA damage in comet assay, but when combined with LPS, it resulted in a significant increase in tail length, tail moment, and olive moment. The data indicate that indoxacarb at 4 mg/kg administered orally for 90 days induced immune-response change. Further, both doses of indoxacarb, when combined with LPS, accelerate immunotoxicity and endotoxin-induced DNA damage.

The mammalian sex-chromosomes (X and Y) have evolved from autosomes and are involved in sex determination and reproductive traits. The Y-chromosome is the smallest chromosome that consists of 2-3% of the haploid genome and may contain between 70 and 200 genes. The Y-chromosome plays major role in male fertility and is suitable to study the evolutionary relics, speciation, and male infertility and/or subfertility due to its unique features such as long non-recombining region, abundance of repetitive sequences, and holandric inheritance pattern. During evolution, many holandric genes were deleted. The current review discusses the mammalian holandric genes and their functions. The commonly encountered infertility and/or subfertility problems due to point or gross mutation (deletion) of the Y-chromosomal genes have also been discussed. For example, loss or microdeletion of sex-determining region, Y-linked gene results in XY males that exhibit female characteristics, deletion of RNA binding motif, Y-encoded in azoospermic factor b region results in the arrest of spermatogenesis at meiosis. The holandric genes have been covered for associating the mutations with male factor infertility.

The wetting behavior of a surface depends on both its surface chemistry and the characteristics of surface morphology and topography. Adding structure to a flat hydrophobic or oleophobic surface increases the effective contact angle and thus the hydrophobicity or oleophobicity of the surface, as exemplified by the lotus leaf analogy. We describe a simple strategy to introduce micropatterned roughness on surfaces of soft materials, utilizing the template of hexagonally packed pores of breath figures as molds. The generated inverse replicas represent micron scale patterned beadlike protrusions on hydrogel surfaces. This added roughness imparts superoleophobic properties (contact angle of the order of 150° and greater) to an inherently oleophobic flat hydrogel surface, when submerged. The introduced pattern on the hydrogel surface changes morphology as it swells in water to resemble morphologies remarkably analogous to the compound eye. Analysis of the wetting behavior using the Cassie-Baxter approximation leads to estimation of the contact angle in the superoleophobic regime and in agreement with the experimental value.

The present work focuses on the in silico characterization of functional divergence of two ovine cathelicidin coding sequence (cds) variants (ie, Cath1 and Cath2) of Indian sheep. Overlapping partial cds of both the cathelicidin variants were cloned in pJet1.2/blunt vector and sequenced. Evolutionary analysis of the Cath2 and Cath1 indicated that the mammalian cathelicidins clustered separately from avian fowlicidins. The avian fowlicidins, which are very different from mammalian cathelicidins (Caths), clearly displayed signatures of purifying selection. The pairwise sequence alignments of translated amino acid sequences of these two sheep cathelicidins showed gaps in the antimicrobial domain of Cath1 variant; however, the amino terminal cathelin regions of both the Caths were conserved. Amino acid sequence analysis of full-length cathelicidins available at public database revealed that Cath1, Cath2, and Cath7 of different ruminant species (including our Cath1 and Cath2 variants) formed individual clads, suggesting that these types have evolved to target specific types of microbes. In silico analysis of Cath1 and Cath2 peptide sequences indicated that the C-terminal antimicrobial peptide domain of Cath2 is more immunogenic than that of the ovine Cath1 due to its higher positive antigenic index, making Cath1 a promising antigen for production of monoclonal antibodies.

This work describes a new method to selectively capture liposomes and other vesicle entities in the patterned pores of breath-figure polymer films. The process involves the deposition of a hydrophobe containing biopolymer in the pores of the breath figure, and the tethering of vesicles to the biopolymer through hydrophobic interactions. The process is versatile, can be scaled up and extended to the deposition of other functional materials in the pores of breath figures.

Dicer, an ribonuclease type III type endonuclease, is the key enzyme involved in biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs), and thus plays a critical role in RNA interference through post transcriptional regulation of gene expression. This enzyme has not been well studied in the Indian water buffalo, an important species known for disease resistance and high milk production. In this study, the primary coding sequence (5,778 bp) of bubaline dicer (GenBank: AB969677.1) was determined and the bubaline Dicer1 biocomputationally characterized to determine the phylogenetic signature among higher eukaryotes. The evolutionary tree revealed that all the transcript variants of Dicer1 belonging to a specific species were within the same node and the sequences belonging to primates, rodents and lagomorphs, avians and reptiles formed independent clusters. The bubaline dicer1 is closely related to that of cattle and other ruminants and significantly divergent from dicer of lower species such as tapeworm, sea urchin and fruit fly. Evolutionary divergence analysis conducted using MEGA6 software indicated that dicer has undergone purifying selection over the time. Seventeen divergent sequences, representing each of the families/taxa were selected to study the specific regions of positive vis-à-vis negative selection using different models like single likelihood ancestor counting, fixed effects likelihood, and random effects likelihood of Datamonkey server. Comparative analysis of the domain structure revealed that Dicer1 is conserved across mammalian species while variation both in terms of length of Dicer enzyme and presence or absence of domain is evident in the lower organisms.

Background: Patients with thalassemia major are largely transfusion dependent and are thus exposed to a variety of risks such as transmission of infectious diseases, iron overload and alloimmunization. This study was performed to determine the prevalence of human immune deficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and red cell antibodies among multiple-transfused thalassemic patients in and around the national capital region., Materials and Methods: The Department of Transfusion Medicine, Indraprastha Apollo Hospitals, conducted this study in collaboration with the National Thalassemia Welfare Society over a period of 1 year starting February2011. Blood samples from the patients were tested for blood group, red cell alloantibody/ies, anti-HIV, anti-HCV and hepatitis B surface antigen (HBsAg) by ELISA and for the respective viral ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) by nucleic acid testing (NAT)., Results: A total of 462 thalassemics which consists of 290 males and 172 females were tested. The overall alloimmunization rate was 4.1% and anti-Kell was the most common antibody identified. Thirteen cases (2.8%) were positive for HBsAg by ELISA, 107 (23.1%) were reactive for anti HCV and 11 (2.38%) for anti HIV antibodies. Further screening and discriminatory assays by NAT confirmed the presence of HBV DNA in 11 cases, HIV RNA in 7 cases and HCV RNA in 48 cases., Conclusion: In spite of advances in Immunohematology and infectious marker testing in recent years, the rates of alloimmunization and infectious marker positivity remains high among multiply transfused patients like thalassemics. Provision of safe and adequate blood supply to these patients is a key to improving their quality-of-life and longevity.

Elevated homocysteine is a risk marker for several major human pathologies. Emerging evidence suggests that perturbations of folate/homocysteine metabolism can directly modify production of inflammatory mediators. Pemetrexed acts by inhibiting thymidylate synthetase (TYMS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). EA.hy 926 cells grown under low ("Lo") and high ("Hi") folate conditions were treated with pemetrexed. The concentrations of several intracellular folate derivatives were measured using LC-MRM/MS. Lo cells had lower total folate concentrations and a different distribution of the intracellular folate derivatives than Hi cells. Treatment with pemetrexed caused a decrease in individual folate analytes. Microarray analysis showed that several genes were significantly up or down-regulated in pemetrexed treated Lo cells. Several of the significantly up-regulated transcripts were inflammatory. Changes in transcript levels of selected targets, including C3, IL-8, and DHFR, were confirmed by quantitative RT-PCR. C3 and IL-8 transcript levels were increased in pemetrexed-treated Lo cells relative to Lo controls; DHFR transcript levels were decreased. In Lo cells, IL-8 and C3 protein concentrations were increased following pemetrexed treatment. Pemetrexed drug treatment was shown in this study to have effects that lead to an increase in pro-inflammatory mediators in Lo cells. No such changes were observed in Hi cells, suggesting that pemetrexed could not modify the inflammatory profile in the context of cellular folate sufficiency., (Copyright © 2012. Published by Elsevier B.V.)

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) is a useful biomarker of oxidative stress. However, its analysis can be challenging because 8-oxo-dGuo must be quantified in the presence of dGuo, without artifactual conversion to 8-oxo-dGuo. Urine is the ideal biological fluid for population studies, because it can be obtained noninvasively and it is less likely that artifactual oxidation of dGuo can occur because of the relatively low amounts that are present compared with hydrolyzed DNA. Stable isotope dilution liquid chromatography-selected reaction monitoring/mass spectrometry (LC-SRM/MS) with 8-oxo-[(15)N(5)]dGuo as internal standard provided the highest possible specificity for 8-oxo-dGuo analysis. Furthermore, artifact formation was determined by addition of [(13)C(10)(15)N(5)]dGuo and monitoring of its conversion to 8-oxo-[(13)C(10)(15)N(5)]dGuo during the analytical procedure. 8-Oxo-dGuo concentrations were normalized for interindividual differences in urine flow by analysis of creatinine using stable isotope dilution LC-SRM/MS. A significant increase in urinary 8-oxo-dGuo was observed in tobacco smokers compared with nonsmokers either using simple urinary concentrations or after normalization for creatinine excretion. The mean levels of 8-oxo-dGuo were 1.65ng/ml and the levels normalized to creatinine were 1.72μg/g creatinine. Therefore, stable isotope dilution LC-SRM/MS analysis of urinary 8-oxo-dGuo complements urinary isoprostane (isoP) analysis for assessing tobacco-smoking-induced oxidative stress. This method will be particularly useful for studies that employ polyunsaturated fatty acids, in which a reduction in arachidonic acid precursor could confound isoP measurements., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Previously, we established that 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (HETE) was a significant cyclooxygenase (COX)-2-derived arachidonic acid (AA) metabolite in epithelial cells. Stable isotope dilution chiral liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization (ECAPCI)/mass spectrometry (MS) was used to quantify COX-2-derived eicosanoids in the human colorectal adenocarcinoma (LoVo) epithelial cell line, which expresses both COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 11(R)-HETE secretion reached peak concentrations within minutes after AA addition before rapidly diminishing, suggesting further metabolism had occurred. Surprisingly, recombinant 15-PGDH, which is normally specific for oxidation of eicosanoid 15(S)-hydroxyl groups, was found to convert 11(R)-HETE to 11-oxo-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (ETE). Furthermore, LoVo cell lysates converted 11(R)-HETE to 11-oxo-ETE and inhibition of 15-PGDH with 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (CAY10397) (50 μM) significantly suppressed endogenous 11-oxo-ETE production with a corresponding increase in 11(R)-HETE. These data confirmed COX-2 and 15-PGDH as enzymes responsible for 11-oxo-ETE biosynthesis. Finally, addition of AA to the LoVo cells resulted in rapid secretion of 11-oxo-ETE into the media, reaching peak levels within 20 min of starting the incubation. This was followed by a sharp decrease in 11-oxo-ETE levels. Glutathione (GSH) S-transferase (GST) was found to metabolize 11-oxo-ETE to the 11-oxo-ETE-GSH (OEG)-adduct in LoVo cells, as confirmed by LC-MS/MS analysis. Bromodeoxyuridine (BrdU)-based cell proliferation assays in human umbilical vein endothelial cells (HUVECs) revealed that the half-maximal inhibitory concentration (IC(50)) of 11-oxo-ETE for inhibition of HUVEC proliferation was 2.1 μM. These results show that 11-oxo-ETE is a novel COX-2/15-PGDH-derived eicosanoid, which inhibits endothelial cell proliferation with a potency that is similar to that observed for 15d-PGJ(2)., (© 2011 American Chemical Society)

Lipid hydroperoxides undergo homolytic decomposition into the bifunctional 4-hydroxy-2( E )-nonenal and 4-oxo-2( E )-nonenal (ONE). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH), deoxyribonucleic acid (DNA) and proteins. Lipid hydroperoxide-derived bifunctional electrophiles are thought to contribute to the pathogenesis of a number of diseases. ONE is an α , β -unsaturated aldehyde that can react in multiple ways and with glutathione, proteins and DNA. Heavy isotope-labeled analogs of ONE are not readily available for conducting mechanistic studies or for use as internal standards in mass spectrometry (MS)-based assays. An efficient onestep cost-effective method has been developed for the preparation of C-9 deuterium-labeled ONE. In addition, a method for specific deuterium labeling of ONE at C-2, C-3 or both C-2 and C-3 has been developed. This latter method involved the selective reduction of an intermediate alkyne either by lithium aluminum hydride or lithium aluminum deuteride and quenching with water or deuterium oxide. The availability of these heavy isotope analogs will be useful as internal standards for quantitative studies employing MS and for conducting mechanistic studies of complex interactions between ONE and DNA bases as well as between ONE and proximal amino acid residues in peptides and proteins.

Glutathione peroxidase-1 (GPX-1) enzyme detoxifies peroxides by reacting with the GSH (reduced glutathione) responsible for the maintenance of the integrity of essential biomolecules. This study was conducted on 100 animals of two Indian draft breeds of cattle (Bos indicus), Nimari, and Malvi. Genomic DNA was isolated from blood samples, and four fragments (80, 71, 78, and 442 bp) of the GPX-1 gene were amplified by polymerase chain reaction. PCR-SSCP analysis in 12% PAGE with silver staining revealed polymorphism in three of the fragments (80, 71, and 442 bp) in these two cattle breeds. Breed differences for the blood biochemical parameters (serum creatine kinase and lactic acid level) and overall draft ability were studied. The genetic polymorphism identified for the GPX-1 gene in this investigation would help in the identification of alleles related to draft capacity in these animals for future genetic improvement.