Enantioselective Synthesis of ß-l-5-[(E)-2-Bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3-(dioxolane-4-yl) Uracil)] (l-BHDU) via Chiral Pure l-Dioxolane.

ß-l-5-((E)-2-Bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3-(dioxolane-4-yl) uracil (l-BHDU, 17) is a potent and selective inhibitor of the varicella-zoster virus (VZV). l-BHDU (17) has demonstrated excellent anti-VZV activity and is a preclinical candidate to treat chickenpox, shingles (herpes zoster), and herpes simplex virus 1 (HSV-1) infections. Its monophosphate prodrug (POM-l-BHDU-MP, 24) demonstrated an enhanced pharmacokinetic and antiviral profile. POM-l-BHDU-MP (24), in vivo, effectively reduced the VZV viral load and was effective for the topical treatment of VZV and HSV-1 infections. Therefore, a viable synthetic procedure for developing POM-l-BHDU-MP (24) is needed. In this article, an efficient approach for the synthesis of l-BHDU (17) from a readily available starting material is described in 7 steps. An efficient and practical methodology for both chiral pure l- & d-dioxolane 11 and 13 were developed via diastereomeric chiral amine salt formation. Neutralization of the amine carboxylate salt of l-dioxolane 10 provides enantiomerically pure l-dioxane 11 (ee ≥ 99%). Optically pure 11 was utilized to construct the final nucleoside l-BHDU (17) and its monophosphate ester prodrug (POM-l-BHDU-MP, 24). Notably, the reported process eliminates expensive chiral chromatography for the synthesis of chiral pure l- & d-dioxolane, which offers avenues for the development and structure-activity relationship studies of l- & d-dioxolane-derived nucleosides.

Synthesis of Fluorinated Nucleosides/Nucleotides and Their Antiviral Properties.

The FDA has approved several drugs based on the fluorinated nucleoside pharmacophore, and numerous drugs are currently in clinical trials. Fluorine-containing nucleos(t)ides offer significant antiviral and anticancer activity. The insertion of a fluorine atom, either in the base or sugar of nucleos(t)ides, alters its electronic and steric parameters and transforms the lipophilicity, pharmacodynamic, and pharmacokinetic properties of these moieties. The fluorine atom restricts the oxidative metabolism of drugs and provides enzymatic metabolic stability towards the glycosidic bond of the nucleos(t)ide. The incorporation of fluorine also demonstrates additional hydrogen bonding interactions in receptors with enhanced biological profiles. The present article discusses the synthetic methodology and antiviral activities of FDA-approved drugs and ongoing fluoro-containing nucleos(t)ide drug candidates in clinical trials.

Synthesis of an Anti-hepatitis B Agent, 2'-Fluoro-6'-methylene-carbocyclic Adenosine (FMCA) and Its Phosphoramidate (FMCAP).

2'-Fluoro-6'-methylene-carbocyclic adenosine (FMCA, 12) and its phosphoramidate prodrug (FMCAP, 14) have been proven as a potential anti-HBV agent against both adefovir-resistant as well as lamivudine-resistant double (rtL180M/rtM204V) mutants. Furthermore, in vitro, these agents have demonstrated significant activity against lamivudine/entecavir triple mutants (L180M + S202G + M204V). These preliminary results encourage us for further biological evaluation of FMCA and FMCAP to develop as a potential clinical candidate as an anti-HBV agent, which may overcome the problem of drug resistance in HBV therapy. To support the preclinical exploration, a scalable synthesis of this molecule was needed. In this communication, a practical and scalable synthesis of FMCA, and its prodrug, is reported via ketone 1. The selective opening of the isopropylidene group of 2 led to compound 3. Protection of the allylic hydroxyl group of 3, followed by fluorination and deprotection, afforded the key intermediate 10, which was condensed with a Boc-protected adenine, followed by deprotection, furnished the target nucleoside FMCA (12) in high yield. Further coupling of phosphorochloridate of L-alanine isopropyl ester (13) with FMCA gave its phosphoramidate prodrug FMCAP (14) in good yield.

2'-Fluoro-6'-methylene carbocyclic adenosine and its phosphoramidate prodrug: A novel anti-HBV agent, active against drug-resistant HBV mutants.

Chronic hepatitis B (CHB) is one of the major causes of morbidity and mortality worldwide. Currently, clinically approved nucleos(t)ide analogs (NAs) are very efficient in reducing the load of hepatitis B virus (HBV) with minimum side effects. However, the long-term administration of antiviral drugs promotes HBV for potential drug resistance. To overcome this problem, combination therapies are administered, but HBV progressively altered mutations remain a threat. Therefore, optimally designed NAs are urgently needed to treat drug-resistant HBV. Herein, 2'-fluoro-6'-methylene carbocyclic adenosine (FMCA) and its phosphoramidate (FMCAP) have been discovered, which may be utilized in combination therapies for curing drug-resistant chronic hepatitis B. In preclinical studies, these carbocyclic NAs demonstrated potential anti-HBV activity against adefovir, as well as lamivudine (LMV/LAM) drug-resistant mutants. In vitro, these molecules have demonstrated significant activity against LMV/entecavir (ETV) triple mutants (L180M + S202G + M204V). Also, preliminary studies of FMCA/FMCAP in chimeric mice and female Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse models having the LMV/ETV triple mutant have shown a high rate of reduction of HBV DNA levels compared to ETV. In this review, we have summarized preclinical studies of FMCA and its phosphoramidate prodrug (FMCAP).

Stereoselective synthesis of 2'-fluoro-6'-methylene carbocyclic adenosine via Vince lactam.

2'-Fluoro-6'-methylene carbocyclic adenosine (FMCA) is a potent and selective inhibitor of wild type as well as drug-resistant hepatitis B virus (HBV) mutants. FMCA demonstrated excellent anti-HBV activity against both adefovir-resistant and lamivudine-resistant double (rtL180M/rtM204V) mutants as well as in lamivudine/entecavir triple mutants (L180M+S202G+M204V) in vitro. Its monophosphate prodrug (FMCAP) demonstrated a greater than 12-fold increase of anti-HBV activity in comparison to that of the nucleoside without elevation of cellular toxicity. In the preliminary in vivo study in chimeric mice harboring the lamivudine/entecavir triple mutant, FMCAP effectively reduced HBV viral load, while entecavir was not effective. Therefore, it was of great interest to develop an efficient synthetic procedure to support the preclinical investigation. In this article, a new approach for the synthesis of FMCA from a readily available starting material (Vince lactam) in 16 steps is described. An efficient and practical methodology for stereospecific preparation of a versatile carbocyclic key intermediate, D-2'-fluoro-6'-methylene cyclopentanol 14, has been developed from diazotization, elimination, stereoselective epoxidation, fluorination, and oxidation-reduction sequence of the Vince lactam in 14 steps. The utility of D-2'-fluoro-6'-methylene cyclopentanol 14 is demonstrated in the preparation of FMCA using the Mitsunobu coupling to introduce the adenine base to synthesize the final nucleoside.

2'-Fluoro-6'-methylene-carbocyclic adenosine phosphoramidate (FMCAP) prodrug: in vitro anti-HBV activity against the lamivudine-entecavir resistant triple mutant and its mechanism of action.

Novel 2'-fluoro-6'-methylene-carbocyclic adenosine (FMCA) monophosphate prodrug (FMCAP) was synthesized and evaluated for its in vitro anti-HBV potency against a lamivudine-entecavir resistant clone (L180M+M204V+S202G). FMCA demonstrated significant antiviral activity against wild-type as well as lamivudine-entecavir resistant triple mutant (L180M+M204V+S202G). The monophosphate prodrug (FMCAP) demonstrated greater than 12-fold (12×) increase in anti-HBV activity without increased cellular toxicity. Mitochondrial and cellular toxicity studies of FMCA indicated that there is no significant toxicity up to 100 µM. Mode of action studies by molecular modeling indicate that the 2'-fluoro moiety by hydrogen bond as well as the Van der Waals interaction of the carbocyclic ring with the phenylalanine moiety of the polymerase promote the positive binding, even in the drug-resistant mutants.

Comparison of Demirjian and Cameriere methods and development of modified Cameriere and Demirjian formula more efficient for North Indian population.

Introduction: Accurate age estimation is of utmost importance in several branches of life, be it disaster victim identification (DVI), sports, fashion, education, and many more. Several studies/formulas have been proposed over the years from various parts of the world and amongst them, Cameriere's method of age estimation is now being accepted globally, and the related work is still one of the most thought about. Aim: The aim of this study was to access the relationship between dental age (DA) and chronological age using Cameriere and Demirjian age estimation method in the north Indian population and develop a population-specific regression formula and validate it in the north Indian population. Materials and Methods: Orthopantomograms (OPG) of 762 children of north India with age groups between 7 and 16 years were collected. Seven left permanent mandibular teeth were analyzed using both Cameriere and Demirjian's age estimation method. The resultant data were subjected to statistical analysis. Results: The mean differences between CAge and DAge with age were 1.21 (males), 0.14 (males) and 1.72 (females), 0.28 (females) respectively, which shows significant disparity, wherein Demirjian follows overestimation and Cameriere follows the underestimation trend. Therefore, we modified these methods using the linear regression model. Conclusion: The modified Demirjian and Cameriere formula after validation shows a better fit in the north Indian state of the Uttar Pradesh population.

Study of Hemato-morphological Features in Neuroblastoma Infiltrating Marrow.

Objective Neuroblastoma typically affects children within the first 5 years of life and accounts for 10% of all pediatric malignancies. Neuroblastoma at onset may manifest as a localized or metastatic illness. The aim of this study was to identify hematomorphological features in neuroblastoma infiltrating marrow as well as to ascertain the prevalence of bone marrow infiltration in neuroblastoma. Materials and Methods This retrospective study included newly diagnosed 79 cases of neuroblastoma, which were referred for bone marrow examination for the staging of the disease. Medical records were retrieved to acquire hematomorphological findings of peripheral blood and bone marrow smears. Statistical Package for Social Sciences, IBM Inc., USA, version 21.0 was used to analyze the data. Results The interquartile age range of neuroblastoma cases was 24.0 to 72.0 months (median = 48 months) with a male to female ratio of 2.7:1. Also, 55.6% (44/79) of cases in the study population showed evidence of marrow infiltration. The bone marrow infiltration was significantly linked to thrombocytopenia ( p = 0.043) and nucleated red blood cells ( p = 0.003) in peripheral blood. The bone marrow smears of cases with infiltration showed a significant shift to the left in the myeloid series ( p = 0.001) and an increased number of erythroid cells ( p = 0.001). Conclusion For neuroblastoma patients, a diligent, exhaustive search for infiltrating cells in bone marrow is advised if thrombocytopenia or nucleated red blood cells are identified on a peripheral blood smear and bone marrow smears showed myeloid left shift with an increased number of erythroid cells.

Early and Effective Diagnosis of Sepsis Using Flow Cytometry.

Objective Sepsis is a major global health issue due to its high death and morbidity rates. To avoid the negative effects of sepsis and decrease mortality, it is vital to diagnose and treat it as soon as possible. Blood cultures can take up to 2 days to give result, and they are not always reliable. According to recent studies, neutrophil CD64 expression might be a sensitive and specific option for assessing sepsis. This study aimed to evaluate the diagnostic performance of a flow cytometry analysis for the expression of neutrophil CD64 in sepsis and its comparison with other standard tests in a tertiary care center. Materials and Methods Prospective analysis on 40 blood samples from suspected sepsis patients admitted to intensive care units with criteria for the systemic inflammatory response syndrome on presentation was performed for expression of neutrophil CD64, C-reactive protein, procalcitonin, and complete blood count. Ten healthy volunteers were also enrolled in this prospective study. The laboratory results were compared in different groups. Results The neutrophil CD64 had the highest diagnostic value to differentiate between patients of sepsis and nonsepsis groups with a sensitivity of 100% (95% confidence interval [CI]: 77.19-100%) and 100% (95% CI: 55.32-86.83%); specificity of 90.00% (95% CI: 59.58-99.49%) and 87.24% (95% CI: 66.69-99.61%); and likelihood ratio of 10.00 and 7.84, respectively. Conclusion The neutrophil CD64 expression provides a more sensitive, specific, and novel marker for the early detection of sepsis in critically ill patients.

Prodrug Strategies for the Development of ß-l-5-((E)-2-Bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3-(dioxolane-4-yl))uracil (l-BHDU) against Varicella Zoster Virus (VZV).

Varicella zoster virus (VZV) establishes lifelong infection after primary disease and can reactivate. Several drugs are approved to treat VZV diseases, but new antivirals with greater potency are needed. Previously, we identified ß-l-5-((E)-2-bromovinyl)-1-((2S,4S)-2-(hydroxymethyl)-1,3-(dioxolane-4-yl))uracil (l-BHDU, 1), which had significant anti-VZV activity. In this communication, we report the synthesis and evaluation of numerous l-BHDU prodrugs: amino acid esters (14-26), phosphoramidates (33-34), long-chain lipids (ODE-l-BHDU-MP, 38, and HDP-l-BHDU-MP, 39), and phosphate ester prodrugs (POM-l-BHDU-MP, 41, and POC-l-BHDU-MP, 47). The amino acid ester l-BHDU prodrugs (l-phenylalanine, 16, and l-valine, 17) had a potent antiviral activity with EC50 values of 0.028 and 0.030 µM, respectively. The phosphate ester prodrugs POM-l-BHDU-MP and POC-l-BHDU-MP had a significant anti-VZV activity with EC50 values of 0.035 and 0.034 µM, respectively, and no cellular toxicity (CC50 > 100 µM) was detected. Out of these prodrugs, ODE-l-BHDU-MP (38) and POM-l-BHDU-MP (41) were selected for further evaluation in future studies.

Hematological Changes in the Second Wave of SARS-CoV-2 in North India.

Background COVID-19 is a rapidly spreading pandemic caused by SARS-CoV-2. India experienced a second wave peak in mid of April 2021, and it emerged as a medical crisis. This study was taken up to show if the hematological and peripheral blood changes can be used as a readily available tool to demarcate the patients needing ICU care so that the ICU can be utilized more prudently.  Material and method One hundred reverse transcription-polymerase chain reaction (RT-PCR) confirmed cases of COVID-19, 50 each from ICU and non-ICU wards, were included in this observational study. At the time of admission blood sample was collected for evaluation of hematological parameters. Results We noted that 74% of patients admitted in ICU were males and 28% were more than 60 years of age. In ICU patients, the absolute neutrophil count (ANC) was significantly raised when compared to non-ICU cases (p=0.023). The nadir absolute lymphocyte count (ALC) was 0.11x109/L in ICU patients and 0.95x109/L in non-ICU patients. There was a significant increase in neutrophil-lymphocyte ratio (NLR; p<0.001) in ICU patients with a proposed cut-off value of 7.73. Platelet-lymphocyte ratio (PLR) was also raised in ICU patients; however, this increase was not significant (p= 0.623). The proposed cut-off value of PLR is 126.73. A significant reduction in a lymphocyte-monocyte ratio (LMR) was observed in ICU patients when compared to non-ICU cases (p<0.001). Thrombocytopenia was more commonly seen in ICU patients; however, this was not statistically significant. Viral-induced cytopathic effects like plasmacytoid lymphocytes with cytoplasmic granules, the presence of toxic changes in neutrophils, and large-sized platelets were commonly observed in ICU patients. Conclusion Our results suggest that hematological parameters like ANC, absolute lymphocyte count (ALC), platelet count, NLR, PLR, and peripheral smear changes are simple assessment factors that can serve as indicators for the severity of COVID-19 and will demarcate the patients who need ICU-care. This will help in the judicious use of ICU facilities for patients who are actually in need.

Hyperlipidemia and Platelet Parameters: Two Sides of the Same Coin.

INTRODUCTION:  Hyperlipidemia is a disorder in which lipid and cholesterol levels in the blood are elevated. Diabetes, coronary heart disease, obesity, and hypertension are commonly linked to hyperlipidemia. Despite this, hyperlipidemia is a widely neglected illness, owing to its asymptomatic nature, ignorance of aberrant lipid profiles on screening, and economic issues in poor countries such as India. Platelets have been shown to have a role in the thrombus consequences of atheromatous damage in hyperlipidemic individuals by initiating and propagating atherosclerotic plaques. Platelets with bigger diameters are thought to be more metabolically, enzymatically, and functionally agile than platelets with lower sizes. In steady-state operation, these bigger platelets release more thromboxane B2 than regular platelets. Platelets with bigger sizes are more hemostatically active and hence have a higher chance of forming a thrombus and thromboembolism. The aim of this study was to compare the values of key platelet parameters and platelet function in hyperlipidemic patients with normal age and sex-matched controls. MATERIAL AND METHODS:  A total of 100 individuals were included in this study, with 68 cases of hyperlipidemia and 32 controls having normal lipid profiles. Platelet volume indices (PVI) such as platelet count (PC), mean platelet volume (MPV), platelet distribution width (PDW), platelet large cell ratio (P-LCR), plateletcrit (PCT), and platelet function (platelet aggregation with adenosine diphosphate, ADP) were compared between hyperlipidemia patients and age sex-matched controls with normal lipid profiles. RESULTS:  The cases had a statistically significant higher mean MPV (10.55 ± 1.81), PDW (14.93 ± 2.82), and P-LCR (30.97 ± 11.74) compared to mean MPV (9.35 ± 1.85), PDW (13.10 ± 2.60), and P-LCR (25.13 ± 12.23) of controls (p-value < 0.05). No significant difference was observed between the study group and control group with respect to mean PC and PCT (p-value > 0.05). In this study, there was a statistically significant increase noted in platelet aggregation percentage in hyperlipidemic patients than in the control group (42.03 ± 25.28 vs 31.25 ± 15.11) (p-value < 0.05). CONCLUSION:  To conclude, platelet parameters are a significant, easy, and cost-effective method for predicting future acute episodes in hyperlipidemic patients that should be utilized more widely. To avoid vascular events, these individuals may require higher antiplatelet dosages and more rigorous hyperlipidemia therapy.

Antiviral activity of novel 2'-fluoro-6'-methylene-carbocyclic adenosine against wild-type and drug-resistant hepatitis B virus mutants.

Novel 2'-fluoro-6'-methylene-carbocyclic adenosine (9) was synthesized and evaluated its anti-HBV activity. The titled compound demonstrated significant antiviral activity against wild-type as well as lamivudine, adefovir and double lamivudine/entecavir resistant mutants. Molecular modeling study indicate that the 2'-fluoro moiety by a hydrogen bond, as well as the van der Waals interaction of the carbocyclic ring with the phenylalanine moiety of the polymerase promote the positive binding, even in the drug resistant mutants.

Oncocytic mucoepidermoid carcinoma (OMEC) of the parotid gland in a 12-year-old patient: A diagnostic challenge on cytology.

Mucoepidermoid carcinoma is the most common malignant salivary gland neoplasm comprising approximately 10% of all tumours of the major salivary gland. Owing to a plethora of morphological variations, it poses a diagnostic challenge on fine-needle aspiration cytology. Oncocytic variant of mucoepidermoid carcinoma is a rare subtype seen in the age group of 20-80 years. It is crucial to make the correct diagnosis on cytology as it has therapeutic implications. Oncocytes can be present in a wide range of salivary gland lesions ranging from non-neoplastic conditions to benign and malignant lesions. We report a case of oncocytic mucoepidermoid carcinoma of the parotid gland in a 12-year-old boy which is the youngest age reported for the same. On cytology, this case was initially diagnosed as Warthin's tumour and was supported by radiology. However, histomorphological findings clinched the diagnosis of an oncocytic variant of mucoepidermoid carcinoma with the aid of immunohistochemistry.

Impact of ongoing COVID-19 pandemic on cytology: An institutional experience.

BACKGROUND: The ongoing COVID-19 pandemic has greatly impacted the health services worldwide, challenging the way modern medicine has been practiced for decades. AIM: The present study documents an institutional experience on its impact on cytology services. MATERIALS & METHODS: The cytology samples received during lock down period in India (24 March to 17 May 2020) were analysis and compared to the samples received during the same time frame in year 2019. RESULTS: The data revealed an overall 92.6% reduction in cytology samples received. All sample types were reduced with a statically significant reduction in thyroid cytology samples (P-value: .023). There was relative increase in breast and lymph node samples; however, this relative increase was not statistically significant. The malignancy rate also significantly increased by 34.1% accompanied by decrease in neoplastic category among the samples received during COVID-19 lockdown period. Breast samples remain the most frequent sample type both in pre-COVID-19 and COVID-19 periods. Majority of fine-needle aspiration done in these cases, during the lockdown period, were either in cases for recurrence or primary diagnosis. CONCLUSION: Prioritization of samples, proper precautions and triaging of patients before procedure helped in carrying out this procedure safely.

Overlapping and distinct functions of two Trichoderma virens MAP kinases in cell-wall integrity, antagonistic properties and repression of conidiation.

We have studied the functions of the Trichoderma virens TmkB, a homologue of the yeast cell-wall integrity MAP kinase Slt2, using gene knockout. The functions of TmkB were compared to those of the pathogenicity MAP kinase homologue (TmkA). Like the tmkA loss-of-function mutants, tmkB mutants exhibited reduced radial growth and constitutive conidiation in dark as well as in liquid shake cultures. The tmkB mutants, in contrast to tmkA mutants, had cell-wall integrity defects, as shown by autolysis of the mycelia and increased sensitivity to cell-wall degrading enzymes. Interestingly, the tmkB mutants were not autolytic on the synthetic Vogels minimal medium. The tmkB mutants had attenuated ability to overgrow the plant pathogen Sclerotium rolfsii, while retaining the ability to overgrow Rhizoctonia solani and Pythium spp., a phenotype also exhibited by the tmkA mutants. This first functional analysis of a cell-wall integrity MAPK in Trichoderma spp., a group of economically important fungi, shows the importance of this signaling pathway in biocontrol. Common phenotypes of the TmkA and TmkB pathways suggest that the two MAPKs may share some substrates, perhaps subunits of key transcription factors, thus dependent on two phosphorylation events for their activity.

Synthesis of 2'-deoxy-2'-fluoro-2'-C-methyl spiro cyclopentyl carbocyclic uridine analog as potential inhibitors of HCV NS5B polymerase.

Synthesis of 1-((4 R,5S,6R,7R)-5,6-dihydroxy-7-(hydroxymethyl)spiro[2.4]heptan-4-yl)pyrimidine-2,4(1H,3H)-dione (12) and its phosphoramidate prodrug 18 is reported. The synthesis of the targeted compound 12 was initiated from triol 1. By the introduction of a substituent methylene group at 6-position of 4, followed by Simmons-Smith cyclopropanation and amination, key intermediate 10 was synthesized. The intermediate amine 10 was utilized to synthesize the nucleoside 12. Furthermore, the nucleoside 12 was derivatized to 2'-α-hydroxy-2'-ß-methyl (23) and 2'-α-fluoro-2'-ß-methyl (27) analogs. All synthesized derivatives of spiro-cyclopropyl carbocyclic uridine analogs 12, 18, 23 and 27 were evaluated for anti-HCV activity, but none of the compounds, reported in this article show any anti-HCV activity.

No yield penalty under favorable conditions paving the way for successful adoption of flood tolerant rice.

Flooding is one of the major constraints for rice production in rainfed lowlands, especially in years and areas of high rainfall. Incorporating the Sub1 (Submergence1) gene into high yielding popular varieties has proven to be the most feasible approach to sustain rice production in submergence-prone areas. Introgression of this QTL into popular varieties has resulted in considerable improvement in yield after flooding. However, its impact under non-flooded conditions or years have not been thoroughly evaluated which is important for the farmers to accept and adopt any new version of their popular varieties. The present study was carried out to evaluate the effect of Sub1 on grain yield of rice in different genetic backgrounds, under non-submergence conditions, over years and locations. The study was carried out using head to head trials in farmer's fields, which enable the farmers to more accurately compare the performance of Sub1 varieties with their recurrent parents under own management. The data generated from different head to head trials revealed that the grain yield of Sub1 varieties was either statistically similar or higher than their non-Sub1 counterparts under non-submergence conditions. Thus, Sub1 rice varieties show no instance of yield penalty of the introgressed gene.

Temperature-Dependent Postmortem Changes in Human Cardiac Troponin-T (cTnT): An Approach in Estimation of Time Since Death.

Estimation of time of death is an indispensible requirement of every medico-legal autopsy, but unfortunately, there is not a single method by which it could be determined accurately. This study focused on the temperature-dependent postmortem degradation of cardiac troponin-T and its association with postmortem interval (PMI) in human. The analysis involved extraction of the protein, separation by denaturing gel electrophoresis (SDS-PAGE), and visualization by Western blot using cTnT-specific monoclonal antibodies. The area of the bands within a lane was quantified by scanning and digitizing the image using Gel Doc (Universal Hood). The results indicate a characteristic banding pattern among human cadavers (n = 6) and a pseudo-linear relationship between percentage of cTnT degradation and the log of the time since death (r > 0.95), which can be used to estimate the postmortem interval. The data presented demonstrate that this technique can provide an extended time range during which PMI can be more accurately estimated.

ß-l-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (l-BHDU) prevents varicella-zoster virus replication in a SCID-Hu mouse model and does not interfere with 5-fluorouracil catabolism.

The alphaherpesvirus varicella-zoster virus (VZV) causes chickenpox and shingles. Current treatments are acyclovir (ACV) and its derivatives, foscarnet and brivudine (BVdU). Additional antiviral compounds with increased potency and specificity are needed to treat VZV, especially to treat post-herpetic neuralgia. We evaluated ß-l-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (l-BHDU, 1) and 5'-O-valyl-l-BHDU (2) in three models of VZV replication: primary human foreskin fibroblasts (HFFs), skin organ culture (SOC) and in SCID-Hu mice with skin xenografts. The efficacy of l-BHDU in vivo and its drug-drug interactions were previously not known. In HFFs, 200µM l-BHDU was noncytotoxic over 3days, and l-BHDU treatment reduced VZV genome copy number and cell to cell spread. The EC50 in HFFs for l-BHDU and valyl-l-BHDU were 0.22 and 0.03µM, respectively. However, l-BHDU antagonized the activity of ACV, BVdU and foscarnet in cultured cells. Given its similar structure to BVdU, we asked if l-BHDU, like BVdU, inhibits 5-fluorouracil catabolism. BALB/c mice were treated with 5-FU alone or in combination with l-BHDU or BVdU. l-BHDU did not interfere with 5-FU catabolism. In SCID-Hu mice implanted with human skin xenografts, l-BHDU and valyl-l-BHDU were superior to ACV and valacyclovir. The maximum concentration (Cmax) levels of l-BHDU were determined in mouse and human tissues at 2h after dosing, and comparison of concentration ratios of tissue to plasma indicated saturation of uptake at the highest dose. For the first time, an l-nucleoside analog, l-BHDU, was found to be effective and well tolerated in mice.