Direct detection of resistance to fluoroquinolones/SLIDs in sputum specimen by GenoType MTBDRsl v.2.0 assay A study from Eastern Uttar Pradesh, India.

BACKGROUND: According to World Health Organization (WHO), drug-resistant tuberculosis (DR-TB) is a major contributor to antimicrobial resistance globally and continues to be a public health threat. Annually, about half a million people fall ill with DR-TB globally. The gradual increase in resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs), poses a serious threat to effective TB control and adequate patient management. Therefore, WHO suggests the use of GenoType MTBDRsl v.2.0 assay for detection of multiple mutations associated with FQs and SLIDs. Hence, the study was conducted to determine the prevalence of resistance to FQs and SLIDs by comparing direct GenoType MTBDRsl v.2.0 assay with phenotypic drug susceptibility testing (DST). METHODS: The study was conducted on 1320 smear positive sputum samples from a total of 2536 RR-TB, confirmed by GeneXpert MTB/RIF. The smear positive specimens were decontaminated, and DNA extraction was performed. Furthermore, the extracted DNA was used for GenoType MTBDRsl v.2.0 assay. While 20% of the decontaminated specimens were inoculated in Mycobacterium growth indicator tube (MGIT) for drug susceptibility testing (DST). RESULTS: Out of 1320 smear positive sputum samples, 1178 were identified as Mycobacterium tuberculosis complex (MTBC) and remaining were negative by GenoType MTBDRsl v.2.0 assay. Of the 1178 MTBC positive, 26.6% were sensitive to both FQs and SLIDs, whereas 57.3% were only FQs resistant and 15.9% were resistant to both FQs and SLIDs. Further DST of 225 isolates by liquid culture showed that 17% were sensitive to both FQs and SLIDs, 61.3% were only FQs resistant and 21.3% were resistant to both. The specificity for FQs and SLIDs was 92.31% and 100% whereas sensitivity was 100% respectively by GenoType MTBDRsl v.2.0 assay in direct sputum samples. CONCLUSIONS: Our study clearly suggests that GenoType MTBDRsl v.2.0 assay is a reliable test for the rapid detection of resistance to second-line drugs after confirmation by GeneXpert MTB/RIF assay for RR-TB. Though, high rate FQ (ofloxacin) resistance was seen in our setting, moxifloxacin could be used as treatment option owing to very low resistance.

Detection of clinically important non tuberculous mycobacteria (NTM) from pulmonary samples through one-step multiplex PCR assay.

BACKGROUND: The burden of non-tuberculous mycobacterial (NTM) disease is increasing worldwide but still its diagnosis is delayed and it is mistaken as multidrug-resistant tuberculosis (MDR-TB).The present study was performed to develop a multiplex PCR assay for detection and identification of clinically most common NTM to the species level from pulmonary samples. RESULTS: Out of 50 isolates, 26 were identified as Mycobacterium kansasii (MK), 20 were identified as Mycobacterium abscessus (MA) and 4 were identified as Mycobacterium avium complex (MAC) through multiplex PCR and further confirmed by sequencing. CONCLUSION: Our study showed that multiplex PCR assay is a simple, convenient, and reliable technique for detection and differential identification of major NTM species.

Purification and characterization of guanylate kinase, a nucleoside monophosphate kinase of Brugia malayi.

Guanylate kinase, a nucleoside monophosphate kinase of Brugia malayi which is involved in reversible transfer of phosphate groups from ATP to GMP, was cloned, expressed and characterized. The native molecular mass of BmGK was found to be 45 kDa as determined by size exclusion chromatography and glutaraldehyde cross-linking which revealed that the protein is homodimer in nature. This is a unique characteristic among known eukaryotic GKs. GMP and ATP served as the most effective phosphate acceptor and donor, respectively. Recombinant BmGK utilized both GMP and dGMP, as substrates showing Km values of 30 and 38 µ m, respectively. Free Mg+2 (un-complexed to ATP) and GTP play a regulatory role in catalysis of BmGK. The enzyme showed higher catalytic efficiency as compared with human GK and showed ternary complex (BmGK-GMP-ATP) formation with sequential substrate binding. The secondary structure of BmGK consisted of 45% α-helices, 18% ß-sheets as revealed by CD analysis. Homology modelling and docking with GMP revealed conserved substrate binding residues with slight differences. Differences in kinetic properties and oligomerization of BmGK compared with human GK can provide the way for design of parasite-specific inhibitors.

A Hospital-Based Cross-Sectional Study on the Histopathology of Upper Gastrointestinal Tract Endoscopic Biopsy in Dyspeptic Patients.

Background Dyspepsia is one of the most common GI complaints encountered in clinical practice. Histopathological assessment of endoscopic gastric mucosa biopsy is crucial to delineate the exact cause of dyspepsia to guide patients' management. Objectives The aim of this study was to determine the histopathological spectrum of upper gastrointestinal (GI) tract endoscopic biopsies and to study the age and sex distribution of the predominant upper GI lesions. Methods A cross-sectional study was conducted in the Department of Pathology, Rajendra Institute of Medical Sciences, Ranchi, Jharkhand, India, from January 2022 to December 2023. All endoscopic mucosal biopsies of the esophagus, stomach, and duodenum (first and second parts) lesions were examined under a microscope for histopathological findings. Results Out of 250 endoscopic biopsies studied, there were 76 cases of esophageal biopsies, 149 cases of gastric biopsies, and 25 cases of duodenal biopsies. The male-to-female ratio was 1.2:1. Non-neoplastic lesions were more common than neoplastic lesions. The most common lesions encountered were esophagitis in the esophagus, gastritis in the stomach, and duodenitis in the duodenum. Conclusion The main organic cause of dyspepsia in our setting was chronic gastritis. We conclude that endoscopy of the upper GI tract and histopathological examination help in the earlier detection of both benign and malignant lesions. This aids in better timely management of the patients and improves the overall treatment provided resulting in a better prognosis.

A 12-week lifestyle intervention for middle-aged, overweight men who are supporters of local sporting clubs.

OBJECTIVE: To evaluate the effectiveness of a 12-week lifestyle program for changes in healthy lifestyle knowledge, health perceptions and body composition of middle-aged, overweight men. METHODS: A participatory, action-based experimental design was employed with a convenience sample (n = 24) of middle-aged men who were supporters of either a local rugby league or rugby union club. Participants attended an introductory session and baseline testing in week one, participated in once-weekly group circuit exercise and lifestyle education sessions for 10 weeks and attended post-testing and project evaluation in week 12. RESULTS: Fourteen participants completed the project. Healthy lifestyle knowledge did not improve significantly. As a combined group there were significant improvements in both physical and mental components of the SF12 questionnaire and in waist girth. The rugby league cohort achieved significant improvement in the SF12 physical component, weight, BMI and waist girth. The rugby union cohort achieved significant improvement in the SF12 mental component and waist girth. Participants reported a variety of health improvement and lifestyle changes following the project and reported appreciation at the involvement of the sporting club. CONCLUSIONS: The men's lifestyle program resulted in significant improvement in body composition, resulting in a reduction in obesity-related disease risk in some participants.

Molecular cloning and characterization of glucose-6-phosphate dehydrogenase from Brugia malayi.

Glucose-6-phosphate dehydrogenase (G6PD), a regulatory enzyme of the pentose phosphate pathway from Brugia malayi, was cloned, expressed and biochemically characterized. The Km values for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) were 0.25 and 0.014 mm respectively. The rBmG6PD exhibited an optimum pH of 8.5 and temperature, 40 °C. Adenosine 5' [γ-thio] triphosphate (ATP-γ-S), adenosine 5' [ß,γ-imido] triphosphate (ATP-ß,γ-NH), adenosine 5' [ß-thio] diphosphate (ADP-ß-S), Na+, K+, Li+ and Cu++ ions were found to be strong inhibitors of rBmG6PD. The rBmG6PD, a tetramer with subunit molecular weight of 75 kDa contains 0.02 mol of SH group per mol of monomer. Blocking the SH group with SH-inhibitors, led to activation of rBmG6PD activity by N-ethylmaleimide. CD analysis indicated that rBmG6PD is composed of 37% α-helices and 26% ß-sheets. The unfolding equilibrium of rBmG6PD with GdmCl/urea showed the triphasic unfolding pattern along with the highly stable intermediate obtained by GdmCl.

Augmented photocatalytic degradation of Acetaminophen using hydrothermally treated g-C3N4 and persulfate under LED irradiation.

Photocatalytic degradation of organic pollutants in water using graphitic carbon nitride and persulfate under visible light (g-C3N4/PS system) has been studied. Here, we demonstrate augmentation of photocatalytic degradation of Acetaminophen (AAP) using hydrothermally treated g-C3N4 and PS under 400 nm LED irradiation (HT-g-C3N4/PS system). A pseudo-first-order rate constant (kobs, 0.328 min-1) for degradation of AAP using HT-g-C3N4/PS system was determined to be 15 times higher compared to g-C3N4/PS system (kobs, 0.022 min-1). HT-g-C3N4 showed a higher surface area (81 m2/g) than g-C3N4 (21 m2/g). Photocurrent response for HT-g-C3N4 was higher (1.5 times) than g-C3N4. Moreover, Nyquist plot semicircle for HT-g-C3N4 was smaller compared to g-C3N4. These results confirm effective photoelectron-hole separation and charge-transfer in HT-g-C3N4 compared to g-C3N4. AAP degradation using HT-g-C3N4/PS system was significantly inhibited with O2.- and h+ scavengers compared to 1O2,SO4.- and HO. scavengers. ESR results revealed O2.- formation in HT-g-C3N4/PS system. Moreover, photocurrent measurements reveal AAP oxidation by h+ of HT-g-C3N4 was effective than g-C3N4. HT-g-C3N4 was reused for five cycles in HT-g-C3N4/PS system. Augmented photocatalytic degradation of AAP by HT-g-C3N4/PS system compared to g-C3N4/PS is attributed to effective photoelectron hole separation of HT-g-C3N4 that generates O2.- and h+ for oxidation of pollutant. Importantly, electrical energy per order (EEO) was 7.2 kWh m-3 order-1. kobs for degradation of AAP in simulated groundwater and tap water were determined as 0.029 and 0.035 min-1, respectively. Degradation intermediates of AAP were proposed. AAP ecotoxicity against marine bacteria Aliivibrio fischeri was completely removed after treatment by HT-g-C3N4/PS system.

Energy efficient photocatalytic activation of peroxymonosulfate by g-C3N4 under 400 nm LED irradiation for degradation of Acid Orange 7.

Photocatalytic activation of peroxymonosulfate (PMS) by graphitic carbon nitride (g-C3N4) is emerging as a sulfate radical anion based advanced oxidation process (S-AOP) for degradation of organic pollutants. Importantly, photocatalytic activation of PMS by g-C3N4 is energy intensive as light irradiation requires high electrical energy. Here, we studied activation of PMS by g-C3N4 under 400 nm light emitting diode (LED) irradiation (g-C3N4/PMS/400-LED system) for degradation of Acid Orange 7 (AO7). LED array having optical emission maximum around 400 nm (FWHM = 16 nm), with electrical input power of 1.54 W (14 V and 110 mA) was used for irradiation. Pseudo-first order rate constant (kobs) value for degradation of AO7 by g-C3N4/PMS/400-LED system was determined to be 0.094 min-1. O2·-, SO4·- were revealed by radical scavenging and ESR investigations. kobs value in simulated ground and real tap water were determined to be 0.068 min-1 and 0.063 min-1, respectively. g-C3N4 was stable, and reused four times without any significant loss in its photocatalytic activity. Importantly, electrical energy per order (EEO) for degradation of AO7 by g-C3N4/PMS/400-LED system was determined to be 24.51 kWh m-3 order-1. In contrast, the EEO value for the degradation of AO7 by g-C3N4 activated PMS under visible light irradiation (>400 nm), using conventional xenon lamp, (g-C3N4/PMS/Vis-Xe system) was found to be very high as 2702 kWh m-3 order-1. Thus, the study highlights, LED irradiation source is promising for the development of energy efficient g-C3N4 photocatalytic activation of PMS for removal of organic pollutants.

Immunoreactivity of Brugia malayi Calreticulin and Its Domains with Sera of Different Categories of Bancroftian Filarial Patients.

PURPOSE: We identified calreticulin in human filaria Brugia malayi (BmCRT) that shares 97% homology with Wuchereria bancrofti calreticulin (WbCRT), but only 56% with human calreticulin. We found that BmCRT binds C1q and prevents complement-mediated parasite death; immunization with BmCRT leads to parasite death in a rodent model of the infection. BmCRT could, therefore, be a potential vaccine candidate. In the present study, we determined the levels of BmCRT-reactive IgG and its isotype in bancroftian filarial subjects. METHODS: Recombinant BmCRT (rBmCRT) was prepared, and the sera of endemic normal subjects (EN), microfilaraemics (Mf+) and chronic amicrofilaraemics (ChMf-) from a bancroftian filaria-endemic area and normal subjects from filaria-non-endemic area (NEN) were probed for IgG and its isotypes reacting with rBmCRT and its domains rN, rP and rC. RESULTS: rBmCRT and its rN domain-reactive IgG levels were high in EN and Mf+ groups; rC domain and rP domain showed moderate and very little reactivity, respectively. NEN sera were non-reactive. Moderate levels of rBmCRT-reactive IgG1, IgG3 and IgG4 in EN and Mf+ groups and low levels of IgG2 in Mf+ were found; IgG1 and IgG3 reactivity was found for rBmCRT and its rN domain only, while IgG4 reactivity was moderate for rN domain and low for rP and rC domains. While IgG reactivity was seen in all the endemic subjects, IgG isotype reactivity was found mostly in EN and Mf+ subjects. CONCLUSIONS: Moderate levels of rBmCRT (and its rN domain)-reactive IgG and its isotypes are present in bancroftian subjects. Preponderance of IgG1 and IgG3 isotypes which bind and activate complement has relevance to vaccine potential of BmCRT.

Systems biology under heat stress in Indian cattle.

Transcriptome profiling of Vrindavani and Tharparkar cattle (n = 5 each) revealed that more numbers of genes were dysregulated in Vrindavani than in Tharparkar. A contrast in gene expression was observed with 18.9 % of upregulated genes in Vrindavani downregulated in Tharparkar and 17.8% upregulated genes in Tharparkar downregulated in Vrindavani. Functional annotation of genes differentially expressed in Tharparkar and Vrindavani revealed that the systems biology in Tharparkar is moving towards counteracting the effects due to heat stress. Unlike Vrindavani, Tharparkar is not only endowed with higher expression of the scavengers (UBE2G1, UBE2S, and UBE2H) of misfolded proteins but also with protectors (VCP, Serp1, and CALR) of naïve unfolded proteins. Further, higher expression of the antioxidants in Tharparkar enables it to cope up with higher levels of free radicals generated as a result of heat stress. In this study, we found relevant genes dysregulated in Tharparkar in the direction that can counter heat stress.

Use of manometry for laryngeal mask airway reduces postoperative pharyngolaryngeal adverse events: a prospective, randomized trial.

BACKGROUND: Adverse events such as pharyngolaryngeal complications are indicators of quality patient care. Use of manometry to limit the laryngeal mask airway (LMA) intracuff pressure is not currently a routine practice. This double-blind randomized trial compared pharyngolaryngeal complications in patients managed with manometers to limit the LMA intracuff pressure (<44 mmHg) with patients under routine care. METHOD: Two hundred consenting patients who underwent ambulatory surgery were randomly allocated to pressure-limiting and routine care groups. Anesthesia was induced with propofol and fentanyl, and maintained with desflurane in air-oxygen. An LMA was inserted, and the cuff was inflated as per usual practice. The patients breathed spontaneously. Research assistants measured the LMA intracuff pressure. In the pressure-limiting group, LMA intracuff pressure was adjusted to less than 44 mmHg. No intervention was performed in the routine care group. Sore throat, dysphonia, and dysphagia were assessed at 1, 2, and 24 h postoperatively. Composite pharyngolaryngeal complications were compared using chi-square test. RESULTS: Baseline demographic data were comparable between groups. Mean LMA intracuff pressure was less in the pressure-limiting group versus the routine care group (40 +/- 6 vs. 114 +/- 57 mmHg, P < 0.001). The incidence of composite pharyngolaryngeal complications was significantly lower in the pressure-limiting group versus the routine care group (13.4 vs. 45.6%, P < 0.001), with a relative risk reduction of 70.6%, and a number needed to treat of three (95% CI 2.2-7.5). CONCLUSION: Reduction of LMA intracuff pressure to less than 44 mmHg lowers the incidence of postoperative pharyngolaryngeal complications. The LMA cuff pressures should be measured routinely using manometry, and deflating the intracuff pressure to less than 44 mmHg should be recommended as anesthetic best practice.

The CD200-CD200R cross-talk helps Leishmania donovani to down regulate macrophage and CD4+CD44+ T cells effector functions in an NFκB independent manner.

The lacuna in the knowledge of immunobiology, especially in visceral infections that are fatal if left untreated, are a major hurdle in getting a vaccine candidate for leishmaniasis. Till date, only a few drugs are available to combat human leishmaniasis and a vaccine candidate either prophylactic or preventive is still awaited. Therefore, identification of host and parasitic factors involved in the regulation of specific immune mechanisms are essentially needed. In this study, we observed that CD200-CD200R immune inhibitory axis regulates host macrophages effectors properties and helps antigen experienced T cells (CD4+CD44+ T cells) to acquire anti-inflammatory cytokines (IL-4, IL-10, TGF-ß, IL-27) producing abilities in an NFkB independent manner. After CD200 blocking the macrophages effectively inhibited proliferation of Leishmania amastigotes and also induced the production of IL-12, IFN-γ, TNF-α and nitric oxide (NOx). Further, the blocking of CD200 signaling also restored macrophages MHC-II expression and helped CD4+CD44+ T cells to produce pro-inflammatory cytokines like IL-2, IL-12 and IFN-γ. The finding of this study suggested the importance of immune inhibitory mechanisms in controlling Leishmania growth and survival and therefore, requires more studies to understand its role in vaccine induced immunity.

Mycobacterium tuberculosis nucleoside diphosphate kinase shows interaction with putative ATP binding cassette (ABC) transporter, Rv1273c.

Protein-protein interactions are crucial for all biological processes. Compiling this network provides many new insights into protein function and gives directions for the development of new drugs targeted to the pathogen. Mycobacterium tuberculosis Nucleoside diphosphate kinase (Mtb Ndk) has been reported to promote survival of mycobacterium within the macrophage and contribute significantly to mycobacterium virulence. Hence, the present study was aimed to identify and characterize the interacting partner for Ndk. The in vitro experiments, pull down and far western blotting have demonstrated that Mtb Ndk interacts with Rv1273c, a probable drug ABC transporter ATP-binding protein annotated to export drugs across the membrane. This observation was further confirmed by molecular docking and dynamic simulations studies. The homology model of Rv1273c was constructed and docked with Mtb Ndk for protein-protein interaction analysis. The critical residues involved at interface of Rv1273c-Ndk interaction were identified. MDS and Principal Component analysis carried out for conformational feasibility and stability concluded that the complex between the two proteins is more stable as compared to apo proteins. Our findings would be expected to improve the dissection of protein-protein interaction network and significantly advance our understanding of tuberculosis infection.Communicated by Ramaswamy H. Sarma.

Rv1273c, an ABC transporter of Mycobacterium tuberculosis promotes mycobacterial intracellular survival within macrophages via modulating the host cell immune response.

Mycobacterium proteins, especially cell wall associated proteins, interact with host macrophage to regulate the functions and cytokine production. So, identification and characterization of such proteins is essential for understanding tuberculosis pathogenesis. The role of the ABC transporter proteins in the pathophysiology and virulence of Mycobacterium tuberculosis is not clearly understood. In the present study, Rv1273c, an ABC transporter, has been expressed in a non-pathogenic and fast growing Mycobacterium smegmatis strain to explore its role in host pathogen interactions. Over expression of Rv1273c resulted in enhanced intracellular survival in macrophage as well as modified cell wall architecture. We found altered colony morphology and cell surface properties that might be linked with remodelling of bacterial cell wall which may help in the intracellular survival of mycobacterium. However, the enhanced intracellular survival was not found to be the consequence of an increased resistance to intracellular stresses. The activation of macrophage by Rv1273c was associated with perturbed cytokine production. Pharmacological inhibition experiment and western immunoblotting suggested that this altered cytokine profile was mediated possibly by NF-kB and p38 pathway in macrophage. Overall, the present findings indicated that Rv1273c enhanced mycobacterium persistence and mediated the evasion of immune responses during infection.

Adsorption of Surface Active Ionic Liquids on Different Rock Types under High Salinity Conditions.

A new class of surface active ionic liquids (SAIL) have been reported to be a greener alternative to the conventional surfactants in enhanced oil recovery (EOR). These SAILs work efficiently under harsh salinity conditions encountered in the reservoir thereby recovering more additional oil during the tertiary oil recovery process. Adsorption mechanism of SAILs on different rock surface is however, not yet reported in the literature. This article highlights adsorption mechanism of three cationic SAILs having different headgroups, viz., imidazolium, pyridinium, pyrrolidinium, on different rock surfaces (crushed natural carbonate rock and crushed sandstone rock). All the SAILs studied here however had the same tail length and same anion (Br-) attached to it. XRD and XPS characterization techniques reveal that the crushed natural carbonate rock contains a substantial amount of silica, thus rendering it a slight negative charge. Static adsorption tests show that the retention efficiency on the natural carbonate type of rock for all the SAILs was lower than the conventional cationic surfactant, CTAB. The adsorption data obtained thereby was examined using four different adsorption isotherm models (Langmuir, Freundlich, Redlich-Peterson, and Sips). Results suggest that Sips adsorption isotherm model can satisfactorily estimate the adsorption of all the surface active agents on the natural carbonate rock. Factors like mineralogical composition of rock surface, presence of divalents, temperature, and structure of surfactants strongly affect the amount of surfactant adsorbed on reservoir rock. In order to evaluate the simultaneous effect all these factors as well as their interdependence on the retention capability of the three SAILs, a design of experiments approach has been employed further in this study. Statistical analysis of the data obtained after performing the full factorial experiments reveal that at high salinity, imidazoluim based SAIL show minimal adsorption on crushed natural carbonate rock at higher temperature. In general, at a given ionic strength, with increasing temperature as the amount of divalent in the aqueous solution increases, the amount of SAIL adsorbed on both the rock types decreases. Electrostatic attraction is the basic mechanism in governing adsorption of SAILs on the two types of rock surfaces. Results presented in this work can be used for EOR schemes.

Neurophysiology of spontaneous facial expressions: II. Motor control of the right and left face is partially independent in adults.

Facial expressions are described traditionally as monolithic or unitary entities. However, humans have the capacity to produce facial blends of emotion in which the upper and lower face simultaneously display different expressions. Recent neuroanatomical studies in monkeys have demonstrated that there are separate cortical motor areas for controlling the upper and lower face in each hemisphere that, presumably, also occur in humans. Using high-speed videography, we began measuring the movement dynamics of spontaneous facial expressions, including facial blends, to develop a more complete understanding of the neurophysiology underlying facial expressions. In our part 1 publication in Cortex (2016), we found that hemispheric motor control of the upper and lower face is overwhelmingly independent; 242 (99%) of the expressions were classified as demonstrating independent hemispheric motor control whereas only 3 (1%) were classified as demonstrating dependent hemispheric motor control. In this companion paper (part 2), 251 unitary facial expressions that occurred on either the upper or lower face were analyzed. 164 (65%) expressions demonstrated dependent hemispheric motor control whereas 87 (35%) expressions demonstrated independent or dual hemispheric motor control, indicating that some expressions represent facial blends of emotion that occur across the vertical facial axis. These findings also support the concepts that 1) spontaneous facial expressions are organized predominantly across the horizontal facial axis and secondarily across the vertical facial axis and 2) facial expressions are complex, multi-component, motoric events. Based on the Emotion-type hypothesis of cerebral lateralization, we propose that facial expressions modulated by a primary-emotional response to an environmental event are initiated by the right hemisphere on the left side of the face whereas facial expressions modulated by a social-emotional response to an environmental event are initiated by the left hemisphere on the right side of the face.

Infections in diabetes mellitus and hyperglycemia.

Infections in diabetes mellitus are relatively more common and serious. Diabetic patients run the risk of acute metabolic decompensation during infections, and conversely patients with metabolic decompensation are at higher risk of certain invasive infections. Tight glycemic control is of paramount importance during acute infected or high stress state. Infections in diabetic patients result in extended hospital stays and additional financial burden. Given the risks of not alleviating the metabolic dysregulation and the benefits of decent glycemic control, it is necessary that besides antimicrobial therapy, equal emphasis be placed on intensified glycemic control.

Monitoring thermal and chemical unfolding of Brugia malayi calreticulin using fluorescence and Circular Dichroism spectroscopy.

Calreticulin of Brugia malayi (BmCRT) play very important role in host-parasite interaction. In previous study it was found that BmCRT is responsible for prevention of host classical complement pathway activation via its interaction with first component C1q of the human host. Therefore, BmCRT is an essential protein for parasite survival and an important drug target to fend filariasis. In the present study, we have carried out a systamatic biophysical characterization of BmCRT protein. Unfolding of BmCRT was found to be non-cooperative two-state process in the presence of both denaturant GdmCl and urea. The results also illustrated that protein lost its 50% activity at 1.5M GdmCl and 3M Urea. Partially unfolded and molten-globule like intermediate state was observed at 0.8 to 1.2M GdmCl while Urea unfolding showed intermediate state at 1.2 to 1.6M. Unfolding pathway monitored with the help of apolar quencher, favor above observations. All of these findings support the presence of detectable intermediate state during unfolding pathway of BmCRT. Furthermore, this study indicates that BmCRT is more stable toward temperature (Tm=65°C), pH and trypsin digestion. These differences in properties as compared to host can be fruitfully utilized for synthesis of compounds effective against the parasite.

Investigating the folding pathway and substrate induced conformational changes in B. malayi Guanylate kinase.

Guanylate kinase is one of the key enzymes in nucleotide biosynthesis. The study highlights the structural and functional properties of Brugia malayi Guanylate kinase (BmGK) in the presence of chemical denaturants. An inactive, partially unfolded, dimeric intermediate was observed at 1-2M urea while GdnCl unfolding showed monomer molten globule like intermediate at 0.8-1.0M. The results also illustrate the protective role of substrates in maintaining the integrity of the enzyme. The thermo stability of protein was found to be significantly enhanced in the presence of the substrates. Furthermore, binding of the substrates, GMP and ATP to BmGK changed its GdnCl induced unfolding pattern. Docking and molecular dynamic simulation performed for native BmGK, BmGK bound to GMP and GMP+ATP showed change in the fluctuation in the region between 130 and 150 residues. Arg134 lost its interaction with GMP and Arg145 interaction shifted to ATP after 40ns simulation upon binding of ATP to BmGK-GMP complex. We, thus, propose the importance of specific rearrangements contributed by binding of substrates which participate in the overall stability of the protein. The work here emphasizes on detailed biophysical characterization of BmGK along with the significant role of substrates in modulating the structural and functional properties of BmGK.