High Replication Rate Research Articles

Are replication rates the same across academic fields? Community forecasts from the DARPA SCORE programme.

The Defense Advanced Research Projects Agency (DARPA) programme ‘Systematizing Confidence in Open Research and Evidence' (SCORE) aims to generate confidence scores for a large number of research claims from empirical studies in the social and behavioural sciences. The confidence scores will provide a quantitative assessment of how likely a claim will hold up in an independent replication. To create the scores, we follow earlier approaches and use prediction markets and surveys to forecast replication outcomes. Based on an initial set of forecasts for the overall replication rate in SCORE and its dependence on the academic discipline and the time of publication, we show that participants expect replication rates to increase over time. Moreover, they expect replication rates to differ between fields, with the highest replication rate in economics (average survey response 58%), and the lowest in psychology and in education (average survey response of 42% for both fields). These results reveal insights into the academic community's views of the replication crisis, including for research fields for which no large-scale replication studies have been undertaken yet.

Replication of 15 loci involved in human plasma protein N-glycosylation in 4802 samples from four cohorts.

Human protein glycosylation is a complex process, and its in vivo regulation is poorly understood. Changes in glycosylation patterns are associated with many human diseases and conditions. Understanding the biological determinants of protein glycome provides a basis for future diagnostic and therapeutic applications. Genome-wide association studies (GWAS) allow to study biology via a hypothesis-free search of loci and genetic variants associated with a trait of interest. Sixteen loci were identified by three previous GWAS of human plasma proteome N-glycosylation. However, the possibility that some of these loci are false positives needs to be eliminated by replication studies, which have been limited so far. Here, we use the largest set of samples so far (4802 individuals) to replicate the previously identified loci. For all but one locus, the expected replication power exceeded 95%. Of the 16 loci reported previously, 15 were replicated in our study. For the remaining locus (near the KREMEN1 gene), the replication power was low, and hence, replication results were inconclusive. The very high replication rate highlights the general robustness of the GWAS findings as well as the high standards adopted by the community that studies genetic regulation of protein glycosylation. The 15 replicated loci present a good target for further functional studies. Among these, eight loci contain genes encoding glycosyltransferases: MGAT5, B3GAT1, FUT8, FUT6, ST6GAL1, B4GALT1, ST3GAL4 and MGAT3. The remaining seven loci offer starting points for further functional follow-up investigation into molecules and mechanisms that regulate human protein N-glycosylation in vivo.

The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA.

Nucleoid-associated proteins (NAPs) are DNA binding proteins critical for the organization and function of the bacterial chromosome. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA.IMPORTANCE Bacteria organize their genetic material in a structure called the nucleoid, which needs to be compact to fit inside the cell and, at the same time, dynamic to allow high rates of replication and transcription. Nucleoid-associated proteins (NAPs) play a pivotal role in this process, so their detailed characterization is crucial for our understanding of DNA organization into bacterial cells. Even though NAPs affect DNA-related processes differently, all of them have to oligomerize and bind DNA for their function. The significance of this study is the identification of structural elements involved in the oligomerization and DNA binding of a newly discovered NAP in C. crescentus and the demonstration that structural elements are conserved in evolutionarily distant and functionally distinct NAPs.

Precise analysis of the effect of basal core promoter/precore mutations on the main phenotype of chronic hepatitis B in mouse models.

High replication and mutation rates of hepatitis B virus (HBV) often lead to reduced or suppressed hepatitis B e antigen expression. The most common mutations are genomic variations in the basal core promoter (BCP) and pre-core (PC) regions. However, the effect of BCP/PC mutations on HBV phenotype in vivo remains unclear. We compared and analyzed BCP/PC mutations and BCP/PC reverse mutations in mouse models. In addition to terminating the expression of HBeAg, BCP/PC mutations also resulted in a significant decrease in HBsAg, HBV DNA, and cccDNA in the early stage, and an obvious increase in serum alanine aminotransferase throughout the transfection period. In both groups, serum HBV DNA was positively correlated with intracellular HBV DNA and cccDNA. Further, we found that interleukin-4 (IL-4) and L-10 levels were significantly lower in the BCP/PC(M) group than in the BCP/PC(R) group at 4 weeks post-injection. However, IL-1β was significantly lower in the BCP/PC(M) group than in the BCP/PC(R) group at 26 weeks post-injection. In summary, we precisely analyzed the effect of BCP/PC mutations on the phenotype in vivo, which is important to evaluating disease progression and treatment responses of variable chronic hepatitis B patients.

Foetal Immune Response Activation and High Replication Rate during Generation of Classical Swine Fever Congenital Infection.

Classical swine fever virus (CSFV) induces trans-placental transmission and congenital viral persistence; however, the available information is not updated. Three groups of sows were infected at mid-gestation with either a high, moderate or low virulence CSFV strains. Foetuses from sows infected with high or low virulence strain were obtained before delivery and piglets from sows infected with the moderate virulence strain were studied for 32 days after birth. The low virulence strain generated lower CSFV RNA load and the lowest proportion of trans-placental transmission. Severe lesions and mummifications were observed in foetuses infected with the high virulence strain. Sows infected with the moderately virulence strain showed stillbirths and mummifications, one of them delivered live piglets, all CSFV persistently infected. Efficient trans-placental transmission was detected in sows infected with the high and moderate virulence strain. The trans-placental transmission occurred before the onset of antibody response, which started at 14 days after infection in these sows and was influenced by replication efficacy of the infecting strain. Fast and solid immunity after sow vaccination is required for prevention of congenital viral persistence. An increase in the CD8+ T-cell subset and IFN-alpha response was found in viremic foetuses, or in those that showed higher viral replication in tissue, showing the CSFV recognition capacity by the foetal immune system after trans-placental infection.

Copy Number and Prevalence of Porcine Endogenous Retroviruses (PERVs) in German Wild Boars.

Porcine endogenous retroviruses (PERVs) are integrated in the genome of pigs and are transmitted like cellular genes from parents to the offspring. Whereas PERV-A and PERV-B are present in all pigs, PERV-C was found to be in many, but not all pigs. When PERV-C is present, recombination with PERV-A may happen and the PERV-A/C recombinants are characterized by a high replication rate. Until now, nothing has been known about the copy number of PERVs in wild boars and little is known about the prevalence of the phylogenetically youngest PERV-C in ancient wild boars. Here we investigated for the first time the copy number of PERVs in different populations of wild boars in and around Berlin using droplet digital PCR. Copy numbers between 3 and 69 per genome have been measured. A lower number but a higher variability was found compared to domestic pigs, including minipigs reported earlier (Fiebig et al., Xenotransplantation, 2018). The wild boar populations differed genetically and had been isolated during the existence of the Berlin wall. Despite this, the variations in copy number were larger in a single population compared to the differences between the populations. PERV-C was found in all 92 analyzed animals. Differences in the copy number of PERV in different organs of a single wild boar indicate that PERVs are also active in wild boars, replicating and infecting new cells as has been shown in domestic pigs.

CNV-BAC: Copy number Variation Detection in Bacterial Circular Genome.

Whole-genome sequencing (WGS) is widely used for copy number variation (CNV) detection. However, for most bacteria, their circular genome structure and high replication rate make reads more enriched near the replication origin. CNV detection based on read depth could be seriously influenced by such replication bias. We show that the replication bias is widespread using ∼200 bacterial WGS data. We develop CNV-BAC (CNV-Bacteria) that can properly normalize the replication bias and other known biases in bacterial WGS data and can accurately detect CNVs. Simulation and real data analysis show that CNV-BAC achieves the best performance in CNV detection compared with available algorithms. CNV-BAC is available at https://github.com/XiDsLab/CNV-BAC. Supplementary data are available at Bioinformatics online.

Targeting replication stress response using polypurine reverse hoogsteen hairpins directed against WEE1 and CHK1 genes in human cancer cells

In response to DNA damage, cell cycle checkpoints produce cell cycle arrest to repair and maintain genomic integrity. Due to the high rates of replication and genetic abnormalities, cancer cells are dependent on replication stress response (RSR) and inhibitors of this pathway are being studied as an anticancer approach. In this direction, we investigated the inhibition of CHK1 and WEE1, key components of RSR, using Polypurine Reverse Hoogsteen hairpins (PPRHs) as gene silencing tool. PPRHs designed against WEE1 or CHK1 reduced the viability of different cancer cell lines and showed an increase of apoptosis in HeLa cells. The effect of the PPRHs on cell viability were dose- and time-dependent in HeLa cells. Both the levels of mRNA and protein for each gene were decreased after treatment with the PPRHs. When analyzing the levels of the two CHK1 mRNA splicing variants, CHK1 and CHK1-S, there was a proportional decrease of the two forms, thus maintaining the same expression ratio. PPRHs targeting WEE1 and CHK1 also proved to disrupt cell cycle after 15 h of treatment. Moreover, PPRHs showed a synergy effect when combined with DNA damaging agents, such as methotrexate or 5-Fluorouracil, widely used in clinical practice. This work validates in vitro the usage of PPRHs as a silencing tool against the RSR genes WEE1 and CHK1 and corroborates the potential of inhibiting these targets as a single agent therapy or in combination with other chemotherapy agents in cancer research.

Mutations and methods of analysis of mutations in Hepatitis B virus.

Immunization programmes against hepatitis-B are being carried out since more than three decades but still HBV is a major public health problem. Hepatitis B virus (HBV) genome consists of circular and partial double stranded DNA. Due to partial double stranded DNA, it uses an RNA intermediate during replication. This replicative strategy of HBV and lack of polymerase proofreading activity give rise to error occurrences comparable to retroviruses. The low fidelity of polymerase, overlapping reading frames and high replication rate produces many non-identical variants at every cycle of replication. Therefore, HBV spreads with mutations and variations. The mutations have been reported both in non-structural as well as structural genes of HBV genome. Recent advances in molecular biology have made easier to analyse these mutations. Hepatitis B antiviral therapy and immunization are all influenced by genetic variability. The analysis and understanding of these mutations are important for therapy against hepatitis B and updating of diagnostic tools. The present review discusses about mutations occurring in whole HBV genome. The mutation occurring both in structural and non-structural genes and non-coding regions have been described in details. It is much more informative because most of literature available, covers only individual gene or DNA regions of HBV.

Molecular and Genomic Characterization of PFAB2: A Non-virulent Bacillus anthracis Strain Isolated from an Indian Hot Spring.

Background Thermophilic bacilli in both aerobic or facultative anaerobic forms have been isolated for over a hundred years from different mesophilic or thermophilic environments as they are potential source of bioactive secondary metabolites. But the taxonomic resolution in the Bacillus genus at species or at strain level is very challenging for the insufficient divergence of the 16S rRNA genes. One such recurring problem is among Bacillus anthracis, B. cereus and B. thuringiensis. The disease-causing B. anthracis strains have their characteristic virulence factors coded in two well-known plasmids, namely pXO1 (toxin genes) and pXO2 (capsule genes).Objective The present study aimed at the molecular and genomic characterization of a recently reported thermophilic and environmental isolate of B. anthracis, strain PFAB2.Methods We performed comparative genomics between the PFAB2 genome and different strains of B. anthracis, along with closely related B. cereus strains.Results The pangenomic analysis suggests that the PFAB2 genome harbors no complete prophage genes. Cluster analysis of Bray-Kurtis similarity resemblance matrix revealed that gene content of PFAB2 is more closely related to other environmental strains of B. anthracis. The secretome analysis and the in vitro and in vivo pathogenesis experiments corroborate the avirulent phenotype of this strain. The most probable explanation for this phenotype is the apparent absence of plasmids harboring genes for capsule biosynthesis and toxins secretion in the draft genome. Additional features of PFAB2 are good spore-forming and germinating capabilities and rapid replication ability.Conclusion The high replication rate in a wide range of temperatures and culture media, the non-pathogenicity, the good spore forming capability and its genomic similarity to the Ames strain together make PFAB2 an interesting model strain for the study of the pathogenic evolution of B. anthracis.

COMMON LAB ABNORMALITIES FROM AN UNCOMMON SOURCE: A CASE REPORT

SESSION TITLE: Wednesday Medical Student/Resident Case Report Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/23/2019 09:45 AM - 10:45 AM INTRODUCTION: Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma (NHL) of B cells. Although its genesis is typically in the lymph nodes, it can form in extra-nodal sites such as the gastrointestinal tract and central nervous system. This disease usually presents with lymphadenopathy, fever, weight loss or fatigue. On occasion, this condition can also present with refractory hypoglycemia and lactic acidosis. CASE PRESENTATION: This is a case of a 66 year old female with a history of diabetes and DLBCL successfully treated with rituximab who presented with lethargy. Initial labs were remarkable for severe hypoglycemia and lactic acidosis of unknown origin. The patient was subsequently intubated, started on broad spectrum antibiotics and admitted to the intensive care unit. Her hypoglycemia was suspected to be secondary to glimepiride toxicity in the setting of acute renal failure and she was started on octreotide. However, her hypoglycemia was refractory to treatment. Her lactic acid continued to rise despite adequate fluid resuscitation and vasopressor support. Exploratory laparotomy was performed out of concern for mesenteric ischemia and revealed a necrotic appendix that was subsequently removed. Surgical pathology was consistent with recurrent DLBCL. Salvage chemotherapy was attempted with improvement in hypoglycemia and lactic acidosis; however, the patient ultimately succumbed to her illness several days later. DISCUSSION: DLBCL typically presents with nodal symptoms, fever, or night sweats. However, there have been a few documented cases of patients initially presenting with refractory hypoglycemia and worsening lactic acidosis despite adequate fluid resuscitation and vasopressor support. These findings presenting as a single entity has been associated with the Warburg effect, meaning that the high rate of replication and glycolysis in cancer cells ultimately leads to increased lactic acid formation and glucose consumption. In the setting of NHL, this is a rare complication and is associated with rapid disease progression and poor outcomes. CONCLUSIONS: This case is an example of an atypical etiology of common lab abnormalities. The patient had both hypoglycemia and an elevated lactate that were unresponsive to traditional therapies. Although a component of the patient's refractory hypoglycemia could have been due to glimepiride, its improvement with chemotherapy is consistent with it being related to DLBCL. In patients with refractory hypoglycemia and rising lactic acidosis with a history of malignancy, relapse should be considered as part of the differential diagnosis. Reference #1: Tanios G, Aranguren IM, Goldstein JS, Patel CB. Diffuse large B-cell lymphoma: A metabolic disorder? The American journal of case reports. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859735/. Published December 2, 2013. Accessed March 13, 2019. Reference #2: Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science (New York, N.Y.). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849637/. Published May 22, 2009. Accessed March 13, 2019. DISCLOSURES: No relevant relationships by Soorya Aggarwal, source=Web Response No relevant relationships by Robert Decker, source=Web Response No relevant relationships by Breanna Goldner, source=Web Response No relevant relationships by Alaynna Kears, source=Web Response

Collisions on the Busy DNA Highway Set Up Barriers for Reprogramming.

Why most cells remain refractory to transcription factor (TF)-induced fate conversion remains largely mysterious, with the answers holding important instructions on how to effectively direct cell identities. In this issue of Cell Stem Cell, Babos etal. (2019) show that conflicts caused by simultaneous high transcription and high replication rates are to blame.

A60 Revealing the evolution of virulence in RNA viruses

A combination of high rates of mutation and replication, coupled with strong natural selection, ensures that RNA viruses experience rapid genotypic and phenotypic evolution. Such a ‘fast-forward’ evolution enables viruses to rapidly adapt to new host species, evade host immune responses, and to develop resistance to anti-viral drugs. Similarly, rapid evolution allows viruses to attain new levels of virulence, defined as the ability to cause severe disease in hosts. We hypothesize that distinct viral groups share genetic determinants that modulate virulence that have been acquired through convergent evolution. Thus, common patterns reflecting changing virulence-related specific viral groups could be detected. The main goals for this project are (1) to understand how genetic and phenotypic diversity can be generated among different viral groups by analyzing the variation patterns and determining the selective forces behind them (impact in viral fitness) and (2) to understand how fixed mutations can modulate virulence within different viral groups by performing comparison of strains with differing virulence within a longitudinal timescale. The subject of the study is key emerging and re-emerging virus families of medical importance. Such groups include: Coronaviridae (severe acute respiratory syndrome and Middle East respiratory syndrome-associated coronaviruses), Picornaviridae (Hepatitis A virus), Flaviviridae (Yellow fever, West Nile, Hepatitis C, Dengue, and Zika viruses), Togaviridae (Rubella and Chikungunya virus), Bornaviridae (Borna-disease virus), Filoviridae (Ebola and Marburg viruses), Paramyxoviridae (Measles, Nipah, and Hendra viruses), Rhabdoviridae (Lyssaviruses), Arenaviridae (Lassa virus), Bunyaviridae (Hanta- and Crimean-Congo hemorrhagic fever viruses), and Orthomyxoviridae (Influenza A viruses). Viral genomes collected at different time points, different hosts (human and their most closely related animal reservoirs) and different locations will be compiled. Extensive molecular evolutionary analyses will be carried out to infer gene expansion/contraction within groups, rates of evolution, and changes in selection pressure, including the detection of positive selected genes and sites (adaptive evolution). Positively selected sites will be mapped onto the viral protein structures to reveal their impact on function, and hence the location of potential virulence determinants. Virulence changes among particular viral strains and types will be defined and measured according to definitions based on an increase in: (1) transmissibility, (2) host tropism, (3) immune evasion, (4) morbidity and mortality, (5) drug resistance, and by the incorporation of epidemiological data to determine whether high or low virulence strains within different hosts and localities are spreading most efficiently in nature.

Enchained growth and cluster dislocation: A possible mechanism for microbiota homeostasis.

Immunoglobulin A is a class of antibodies produced by the adaptive immune system and secreted into the gut lumen to fight pathogenic bacteria. We recently demonstrated that the main physical effect of these antibodies is to enchain daughter bacteria, i.e. to cross-link bacteria into clusters as they divide, preventing them from interacting with epithelial cells, thus protecting the host. These links between bacteria may break over time. We study several models using analytical and numerical calculations. We obtain the resulting distribution of chain sizes, that we compare with experimental data. We study the rate of increase in the number of free bacteria as a function of the replication rate of bacteria. Our models show robustly that at higher replication rates, bacteria replicate before the link between daughter bacteria breaks, leading to growing cluster sizes. On the contrary at low growth rates two daughter bacteria have a high probability to break apart. Thus the gut could produce IgA against all the bacteria it has encountered, but the most affected bacteria would be the fast replicating ones, that are more likely to destabilize the microbiota. Linking the effect of the immune effectors (here the clustering) with a property directly relevant to the potential bacterial pathogeneicity (here the replication rate) could avoid to make complex decisions about which bacteria to produce effectors against.

Counteraction of HCV-Induced Oxidative Stress Concurs to Establish Chronic Infection in Liver Cell Cultures.

Hepatitis C virus (HCV) is a blood-borne pathogen causing acute and chronic hepatitis. A significant number of people chronically infected with HCV develop cirrhosis and/or liver cancer. The pathophysiologic mechanisms of hepatocyte damage associated with chronic HCV infection are not fully understood yet, mainly due to the lack of an in vitro system able to recapitulate the stages of infection in vivo. Several studies underline that HCV virus replication depends on redox-sensitive cellular pathways; in addition, it is known that virus itself induces alterations of the cellular redox state. However, the exact interplay between HCV replication and oxidative stress has not been elucidated. In particular, the role of reduced glutathione (GSH) in HCV replication and infection is still not clear. We set up an in vitro system, based on low m.o.i. of Huh7.5 cell line with a HCV infectious clone (J6/JFH1), that reproduced the acute and persistent phases of HCV infection up to 76 days of culture. We demonstrated that the acute phase of HCV infection is characterized by the elevated levels of reactive oxygen species (ROS) associated in part with an increase of NADPH-oxidase transcripts and activity and a depletion of GSH accompanied by high rates of viral replication and apoptotic cell death. Conversely, the chronic phase is characterized by a reestablishment of reduced environment due to a decreased ROS production and increased GSH content in infected cells that might concur to the establishment of viral persistence. Treatment with the prooxidant auranofin of the persistently infected cultures induced the increase of viral RNA titer, suggesting that a prooxidant state could favor the reactivation of HCV viral replication that in turn caused cell damage and death. Our results suggest that targeting the redox-sensitive host-cells pathways essential for viral replication and/or persistence may represent a promising option for contrasting HCV infection.

A robust pipeline with high replication rate for detection of somatic variants in the adaptive immune system as a source of common genetic variation in autoimmune disease.

The role of somatic variants in diseases beyond cancer is increasingly being recognized, with potential roles in autoinflammatory and autoimmune diseases. However, as mutation rates and allele fractions are lower, studies in these diseases are substantially less tolerant of false positives, and bio-informatics algorithms require high replication rates. We developed a pipeline combining two variant callers, MuTect2 and VarScan2, with technical filtering and prioritization. Our pipeline detects somatic variants with allele fractions as low as 0.5% and achieves a replication rate of >55%. Validation in an independent data set demonstrates excellent performance (sensitivity > 57%, specificity > 98%, replication rate > 80%). We applied this pipeline to the autoimmune disease multiple sclerosis (MS) as a proof-of-principle. We demonstrate that 60% of MS patients carry 2–10 exonic somatic variants in their peripheral blood T and B cells, with the vast majority (80%) occurring in T cells and variants persisting over time. Synonymous variants significantly co-occur with non-synonymous variants. Systematic characterization indicates somatic variants are enriched for being novel or very rare in public databases of germline variants and trend towards being more damaging and conserved, as reflected by higher phred-scaled combined annotation-dependent depletion (CADD) and genomic evolutionary rate profiling (GERP) scores. Our pipeline and proof-of-principle now warrant further investigation of common somatic genetic variation on top of inherited genetic variation in the context of autoimmune disease, where it may offer subtle survival advantages to immune cells and contribute to the capacity of these cells to participate in the autoimmune reaction.

Breaking Bad: How Viruses Subvert the Cell Cycle.

Interactions between the host and viruses during the course of their co-evolution have not only shaped cellular function and the immune system, but also the counter measures employed by viruses. Relatively small genomes and high replication rates allow viruses to accumulate mutations and continuously present the host with new challenges. It is therefore, no surprise that they either escape detection or modulate host physiology, often by redirecting normal cellular pathways to their own advantage. Viruses utilize a diverse array of strategies and molecular targets to subvert host cellular processes, while evading detection. These include cell-cycle regulation, major histocompatibility complex-restricted antigen presentation, intracellular protein transport, apoptosis, cytokine-mediated signaling, and humoral immune responses. Moreover, viruses routinely manipulate the host cell cycle to create a favorable environment for replication, largely by deregulating cell cycle checkpoints. This review focuses on our current understanding of the molecular aspects of cell cycle regulation that are often targeted by viruses. Further study of their interactions should provide fundamental insights into cell cycle regulation and improve our ability to exploit these viruses.

Enzymes of pyrimidine salvage pathways in intraerythrocytic Plasmodium falciparum.

Malaria remains a significant public health problem worldwide with an estimated annual global incidence of 200 million and an estimated 450,000 annual deaths. Among the five known human malarial species, Plasmodium falciparum is the deadliest and most resistant to antimalarials. Hence, there is a need for new antimalarial targets. The rational design of a drug is usually based on biochemical and physiological differences between pathogens and their hosts. In view of their high rate of replication, parasites require very active nucleic acid synthesis which necessitates large supplies of the indispensable pyrimidine nucleotides. Consequently, delineation of P. falciparum pyrimidine metabolic pathways may reveal potential targets for the chemotherapy of malaria. Previous studies reported the existence of pyrimidine de novo pathways in this organism. The present results demonstrate the presence of enzymes of the pyrimidine salvage pathways in P. falciparum and indicate that this parasite is capable of pyrimidine salvage. Furthermore, some of the pyrimidine salvage enzymes, e.g., dTMP kinase, phosphoribosyltransferase, and uridine phosphorylase could be excellent targets for chemotherapeutic intervention against this parasite.

Diffuse Large B-cell Lymphoma - A Rare Cause of Acute Liver Failure

Acute liver failure is a rare but life-threatening illness with an incidence of 2-8 per million populations. Most common causes of ALF include drug-induced liver injury, viral hepatitis, autoimmune disease, metabolic diseases and very rare malignancy. Malignant infiltration of the liver is a rare presentation of acute liver failure associated with poor prognosis. We report a case of a patient with acute liver failure caused by malignant infiltration by diffuse large B-cell lymphoma. A 28-year-old female with no significant past medical history or social history presented with abdominal distention and jaundice for four days associated with increased fatigue. She denied weight loss, fever, or night sweats. On physical examination, the patient had scleral icterus, distended non-tender abdomen with massive hepatosplenomegaly and minimal hepatic encephalopathy. Initial laboratory work was notable for; ALT 53 IU/L, AST 394 IU/L, Alkaline Phosphatase 433IU/L, Direct Bilirubin 7.4mg/dl, total Bilirubin 11.4mg/dl, PT 17.6secs, INR 1.6, PTT 28.2 secs. Abdominal Ultrasound showed an enlarged diffusely, heterogeneous liver concerning for metastasis or primary hepatic malignancy. An abdominal Computed Tomography scan revealed massive hepatosplenomegaly, heterogeneous hepatic involvement, with lymphomatous involvement of abdominal, pelvic and inguinal lymph nodes. Workup for his acute liver failure including viral, autoimmune and metabolic disease was unrevealing. Patient subsequently underwent an inguinal core lymph node biopsy that showed high-grade lymphoma (CD10-, MUM1-), double expression (IHC +, C MYC, BCL6), high replication rate- Ki-67 80-90%, IPI 3, 85% CD5+, CD10+ and CD 20+, as well as weakly positive for CD19 (Figure 1). The patient was started on chemotherapy, which consisted of hyper CVAD (Vincristine, Rituximab, Cyclophosphamide and Dexamethasone. Initial treatment was well tolerated and the patient was discharged for out-patient continuation of chemotherapy. Acute liver failure is characterized by the development of severe liver injury with impaired synthetic capacity and encephalopathy in patients with a previously normal liver. Acute liver failure due to malignant infiltration is very rare, this case highlights a high index of suspicion, particularly if no obvious etiology identified. Malignancy causing acute hepatic failure must be recognized to avoid inappropriate referral for transplantation and delay in initiating treatment.2342_A Figure 1. Inguinal core lymph node biopsy showing high-grade B-cell lymphoma IHC positive for C-MYC and BCL6.2342_B Figure 2. Axial CT abdomen with massive hepatomegaly (red arrow) and splenomegaly (blue arrow)2342_C Figure 3. Coronal CT abdomen with massive hepatomegaly (red arrow) and splenomegaly (blue arrow)

Focus on recently developed assays for detection of resistance/sensitivity to reverse transcriptase inhibitors.

The biology of HIV is rather complex due to high rate of replication, frequent recombination, and introduction of mutations. This gives rise to a number of distinct variants referred as quasispecies. In addition, the latency within reservoir allows the periodic reactivation of virus replication. The rapid replication of HIV allows immune response escape and establishment of resistance to therapy that can be acquired through drug selection and/or transmitted among individuals. This prompted, over the years, the development of a range of assays aimed to determine drug resistance and sensitivity, to be used both in clinical practice and in antiviral research. Reverse transcriptase (RT) inhibitors have an eminent place among the anti-HIV drugs, being constantly present from the beginning until today in the most commonly used antiviral regimens. This mini-review seeks to provide an up-to-date overview of recent efforts in developing even more reliable and simple methods, of both genotypic and phenotypic types, for specifically detecting drug resistance and sensitivity to RT inhibitors.