Biophysical Characterization of the Membrane Binding Domain of the Pro-apoptotic Protein Bax

The BCL-2 family of proteins tightly regulates the delicate balance between life and death. The pore forming Bax is a pro-apoptotic member belonging to this protein family. At the onset of apoptosis, monomeric cytoplasmic Bax translocates to the outer mitochondrial membrane, forms oligomeric pores thereby letting mitochondrial cytochrome c enter the cytosol and initiate the apoptotic cascade. The C-terminal helix 9 is thought to mediate the membrane binding of BAX. A 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with Glu (charge reversal mutant, EE) or Leu (charge neutralization mutant, LL) have been studied to elucidate the pore formation capabilities of Bax C-terminus and the underlying molecular mechanism. Interactions of the wild-type and the two mutant peptides with zwitterionic and anionic phospholipid membranes caused efficient membrane permeabilization, as documented by release of vesicle-entrapped fluorescent indicator calcein. Light scattering experiments showed that vesicles maintained their integrity upon peptide binding, indicating that the content leakage was due to pore formation and not vesicle degradation. Kinetics of calcein release at various peptide concentrations were used to determine the peptide-peptide association constants and the oligomeric state of the pore. The structure of membrane-bound peptides was analyzed by circular dichroism (CD) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. CD data indicated all three peptides reconstituted in lipid vesicles contained [alpha]-helical and [beta]-strand structures. ATR-FTIR experiments indicated that the minimally hydrated samples of peptides in stacked lipid bilayers (absence of bulk water) were mostly [alpha]-helical but adopted mostly [beta]-sheet conformation in the presence of excess water. Finally, the depth of membrane insertion of the peptides was analyzed using tryptophan fluorescence quenching by dibromo-phosphatidylcholines brominated at various positions of their acyl chains. In case of zwitterionc phospholipid membranes, the single Trp¹⁶ was located at ~9 Å from membrane center. In case of membranes containing 30% of an anionic phospholipid, the depth of membrane insertion of the EE mutant was not affected but the wild-type and the LL mutant peptides were embedded much deeper into the membrane, with Trp¹⁶ located at 3-4 Å from membrane center. These results will help achieve a better understanding of the molecular mechanism of membrane pore formation of Bax protein. In addition, they provide insight into the molecular details of membrane pore formation by peptides and could facilitate the design and production of cytotoxic peptides with improved capabilities to lyse cells such as bacteria or cancer cells

Learning-based inductive invariant synthesis

The problem of synthesizing adequate inductive invariants to prove a program correct lies at the heart of automated program verification. We investigate, herein, learning approaches to synthesize inductive invariants of sequential programs towards automatically verifying them. To this end, we identify that prior learning approaches were unduly influenced by traditional machine learning models that learned concepts from positive and negative counterexamples. We argue that these models are not robust for invariant synthesis and, consequently, introduce ICE, a robust learning paradigm for synthesizing invariants that learns using positive, negative and implication counterexamples, and show that it admits honest teachers and strongly convergent mechanisms for invariant synthesis. We develop the first learning algorithms in this model with implication counterexamples for two domains, one for learning arbitrary Boolean combinations of numerical invariants over scalar variables and one for quantified invariants of linear data-structures including arrays and dynamic lists. We implement the ICE learners and an appropriate teacher, and show that the resulting invariant synthesis is robust, practical, convergent, and efficient. In order to deductively verify shared-memory concurrent programs, we present a sequentialization result and show that synthesizing rely-guarantee annotations for them can be reduced to invariant synthesis for sequential programs. Further, for verifying asynchronous event-driven systems, we develop a new invariant synthesis technique that constructs almost-synchronous invariants over concrete system configurations. These invariants, for most systems, are finitely representable, and can be thereby constructed, including for the USB driver that ships with Microsoft Windows phone

To Study The Efficacy Of Reciprocating Single File System And Multifile Rotary Systems In Elimination Of Root Canal Bacteria And Endotoxins

AIM: To study the efficacy of reciprocating single file system and multifile rotary systems in elimination of root canal bacteria and endotoxins. MATERIAL AND METHOD: Forty permanent single rooted mandibular premolars with straight canals inoculated with Escherichia Coli suspension for 21 days were selected for the study. Teeth were randomly divided into four groups (n=10) according to instrumentation system: Grp. A–Reciproc (VDW); Grp. B–WaveOne (Dentsply Maillefer); Grp. C–MTwo (VDW); and Grp. D–K3( Sybron Endo); Bacterial and endotoxin samples were collected with a sterile paper point before instrumentation  and after instrumentation. Culture methods estimated the colony-forming units (CFU) and the Limulus Amebocyte Lysate test was used for quantification of endotoxins. Results so obtained were calculated and statistically analysed.  RESULT: Results at S1 concluded that bacteria and endotoxins were found in all of the investigated root canals. After instrumentation all systems were associated with the significant reduction of the bacterial load and endotoxins respectively: Grp. A– Reciproc (88.25% and 89.10%); Grp. B– WaveOne (83.53% and 80.59%); Grp. C– MTwo(79.41% and 75.84%) and Grp. D– K3 (67% and 74.4%). Statistically no difference was found amongst the instrumentation systems regarding bacteria and endotoxin elimination (P <0.05).  CONCLUSION: The reciprocating single file, Reciproc and WaveOne were as effective as the multifile rotary systems for the eradication of bacteria and endotoxins from root canals

Mediastinal germ cell tumour masquerading as loculated pleural effusion

Benign mediastinal teratomas often discovered while patients are still asymptomatic. Almost all arise in the anterosuperior mediastinal compartment. Most symptoms result from compression of adjacent structures. We report a case of a large teratoma arising from the anterior mediastinum that presented a confusing clinical picture of loculated pleural effusion which was successfully diagnosed and treated by surgical excisio

Clinical Correlates of Hepatitis B or Hepatitis C Coinfections in People Living with HIV/AIDS (PLHIV)

Introduction: Hepatitis B virus (HBV) coinfected HIV patients are likely to have chronic hepatitis B infection and associated severe liver disease, however effect of hepatitis B on HIV has not been proven to be off any effect. Similarly in HIV/HCV co-infection majority of the studies have shown no significant influenceof hepatitis C on the course of HIV infection, although some studies have demonstrated an association between HCV infection and faster HIV disease progression.14,15 Therefore, further studies are needed to study the impact of HBV/HCV co-infection on course of HIV, specially, in India.Aims and Objectives: To study the clinical, biochemical and immunological profile of PLHIV co-infected with either hepatitis B or hepatitis C virus, the severity of liver disease and hepatitis B and hepatitis C viral loads in these co-infected PLHIV and the association of WHO stage of HIV and immunosuppression withhepatitis B and hepatitis C viral loads as well as severity of liver disease.Method: It was an observational cross-sectional study, involving 30 PLHIV co-infected with either hepatitis B or C. A detailed history and physical examination was done. Complete Haemogram, Liver function tests, kidney function tests, Ultrasonography abdomen, CD4 cell counts, hepatitis B surface antigen (HBsAg),hepatitis B envelope antigen (HBeAg), hepatitis B Viral DNA (HBV DNA) and HCV RNA levels were done. Severity of liver disease was assessed by FIB 4 SCORE.Results: Among the 30 PLHIV subjects 30% were co-infected with HCV 70% were co-infected with HBV (HBsAg positive). All the subjects were asymptomatic for their liver disease. All the subjects were on Anti-Retroviral Therapy (ART) and 80% were in Early WHO stage (T1 and T2) and 20% were in Advanced WHO stage (T3 and T4). It was similar in both HBV and HCV co-infected group. The mean CD4 count of the subjects was 416.70±189.50 cells/mm3 with the range of 69 – 909 cells/mm3. Five subjects (16.67%) had a CD4 count 3.25). In HCV co-infected subjects 3 of 9 (33.33%) had severe liver fibrosis and only 1 of 21 (4.7%) among HBV co-infected had severe liver fibrosis.Among the 9 HCV co-infected subjects, 3 (33.33%) had undetectable HCV RNA. More number of subjects with detectable hepatitis C viral load had severe liver disease as compared to undetectable viral load.In HIV and HBV co-infected subjects the HBeAg positivity was seen in 42.86% subjects and 38.1% subjects had detectable HBV DNA load. Significant correlation was seen between HBeAg positivity and HBV DNA load. No correlation could be found between FIB 4 score and hepatitis B envelope antigen (HBeAg) positivity or HBV DNA load.No correlation between severity of liver disease (FIB 4) score and WHO staging or CD4 count could be seen. WHO staging and CD4 count also did not correlated with HCV RNA load, HBeAg positivity and HBV DNA load.Conclusions: There is no correlation of CD4 count and WHO stage with liver disease severity or hepatitis viral load in patients on HAART. In HIV and HBV co-infected patients high prevalence of HBeAg positivity is seen. Thus it becomes important to look for deranged liver enzymes and HBeAg positivity in PLHIV coinfected with hepatitis B so that ART can be initiated in these patients irrespective of CD4 count. Hepatitis C co-infected subjects are more likely to have severe liver disease inspite of good CD4 count, so specific treatment for hepatitis C virus should be considered