ZBP1 promotes fungi-induced inflammasome activation and pyroptosis,apoptosis, and necroptosis (PANoptosis)

Candida albicans and Aspergillus fumigatus are dangerous fungal pathogens with high morbidity and mortality, particularly in immunocompromised patients. Innate immune-mediated programmed cell death (pyroptosis, apoptosis, necroptosis) is an integral part of host defense against pathogens. Inflammasomes, which are canonically formed upstream of pyroptosis, have been characterized as key mediators of fungal sensing and drivers of proinflammatory responses. However, the specific cell death pathways and key upstream sensors activated in the context of Candida and Aspergillus infections are unknown. Here, we report that C. albicans and A. fumigatus infection induced inflammatory programmed cell death in the form of pyroptosis, apoptosis, and necroptosis (PANoptosis). Further, we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as the apical sensor of fungal infection responsible for activating the inflammasome/pyroptosis, apoptosis, and necroptosis. The Zα2 domain of ZBP1 was required to promote this inflammasome activation and PANoptosis. Overall, our results demonstrate that C. albicans and A. fumigatus induce PANoptosis and that ZBP1 plays a vital role in inflammasome activation and PANoptosis in response to fungal pathogens.     

Benzoic acid production via cascade biotransformation and coupled fermentation-biotransformation

As an important bulk chemical, benzoic acid is currently manufactured from nonrenewable feedstocks under harsh conditions. Although there are natural pathways for biosynthesis of benzoic acid, they are often inefficient and subjected to complex regulation. Here we develop a nonnatural enzyme cascade to efficiently produce benzoic acid from styrene or biogenic L -phenylalanine under mild conditions. By using a modular approach, two whole-cell catalysts Escherichia coli LZ305 and LZ325 are engineered for coexpressing seven and nine enzymes for production of 133–146 mM benzoic acid (16.2–17.8 g/L_aq) with 88–97% conversion via seven- and nine-step cascade biotransformation of styrene and L -phenylalanine, respectively. The seven-step cascade represents a formal high-yielding biocatalytic oxidative cleavage of styrene, and the nine-step cascade showcases the high efficiency of extended nonnatural enzyme cascades. Moreover, to achieve benzoic acid production directly from low-cost renewable glycerol, a novel coupled fermentation-biotransformation process was developed by integration of fermentative production of L -phenylalanine with in situ biotransformation to give 63–70 mM benzoic acid (7.6–8.6 g/L_aq), which is around 20 times higher than the reported value via a natural pathway. The coupled fermentation-biotransformation process could be generally applicable to microbial production of growth-inhibitory or toxic chemicals in high concentrations.     

Identification and characterization of a hitherto unknown nucleotide-binding domain and an intricate interdomain regulation in HflX-a ribosome binding GTPase

A role for HflX in 50S-biogenesis was suggested based on its similarity to other GTPases involved in this process. It possesses a G-domain, flanked by uncharacterized N- and C-terminal domains. Intriguingly, Escherichia coli HflX was shown to hydrolyze both GTP and adenosine triphosphate (ATP), and it was unclear whether G-domain alone would explain ATP hydrolysis too. Here, based on structural bioinformatics analysis, we suspected the possible existence of an additional nucleotide-binding domain (ND1) at the N-terminus. Biochemical studies affirm that this domain is capable of hydrolyzing ATP and GTP. Surprisingly, not only ND1 but also the G-domain (ND2) can hydrolyze GTP and ATP too. Further; we recognize that ND1 and ND2 influence each other’s hydrolysis activities via two salt bridges, i.e. E29-R257 and Q28-N207. It appears that the salt bridges are important in clamping the two NTPase domains together; disrupting these unfastens ND1 and ND2 and invokes domain movements. Kinetic studies suggest an important but complex regulation of the hydrolysis activities of ND1 and ND2. Overall, we identify, two separate nucleotide-binding domains possessing both ATP and GTP hydrolysis activities, coupled with an intricate inter-domain regulation for Escherichia coli HflX.     

InDiaMed: A Comprehensive Database of Indian Medicinal plants for Diabetes

According to International Diabetes Federation (IDF), India has 62.4 million people with diabetes and by 2030 it is predicted that the number will rise to 100 million. Studies claim that there are around 410 experimentally proven Indian medicinal plants which have anti-diabetic activity, of which the mechanism of action of 109 plants has been elucidated or reported. So, the need of the hour is to explore the claims of Indian medicinal flora and open up the facets of many Indian plants which are being examined for their beneficial role in diabetes. So, we created a database (InDiaMed) of Indian medicinal plants that captures their role in anti-diabetic activity. InDiaMed''s features include chemical, pharmacological, biochemical and geographical information of the medicinal plant, scientifically relevant information of the plant, and the coherent research done on it in the field of diabetes. The database also includes the list of poly-herbal formulations which are used for treatment of diabetes in India.

Availability

http://www.indiamed.info     

Avidity‐mediated virus separation using a hyperthermophilic affinity ligand

Immunoaffinity separation of large multivalent species such as viruses is limited by the stringent elution conditions necessary to overcome their strong and highly avid interaction with immobilized affinity ligands on the capture surface. Here we present an alternate strategy that harnesses the avidity effect to overcome this limitation. Red clover necrotic mosaic virus (RCNMV), a plant virus relevant to drug delivery applications, was chosen as a model target for this study. An RCNMV binding protein (RBP) with modest binding affinity (K_D ～100 nM) was generated through mutagenesis of the Sso7d protein from Sulfolobus solfataricus and used as the affinity ligand. In our separation scheme, RCNMV is captured by a highly avid interaction with RBP immobilized on a nickel surface through a hexahistidine (6xHis) tag. Subsequently, disruption of the multivalent interaction and release of RCNMV is achieved by elution of RBP from the nickel surface. Finally, RCNMV is separated from RBP by exploiting the large difference in their molecular weights (～8 MDa vs. ～10 kDa). Our strategy not only eliminates the need for harsh elution conditions, but also bypasses chemical conjugation of the affinity ligand to the capture surface. Stable non‐antibody affinity ligands to a wide spectrum of targets can be generated through mutagenesis of Sso7d and other hyperthermophilic proteins. Therefore, our approach may be broadly relevant to cases where capture of large multivalent species from complex mixtures and subsequent release without the use of harsh elution conditions is necessary. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Risk Factors for Hyperglycaemia in Pregnancy in Tamil Nadu,India

Introduction

Hyperglycaemia in pregnancy (HIP), i.e. gestational diabetes mellitus (GDM) and diabetes in pregnancy (DIP), increases the risk of various short- and long-term adverse outcomes. However, much remains to be understood about the role of different risk factors in development of HIP.

Objective

The aims of this observational study were to examine the role of potential risk factors for HIP, and to investigate whether any single or accumulated risk factor(s) could be used to predict HIP among women attending GDM screening at three centres in urban, semi-urban and rural Tamil Nadu, India.

Methodology

Pregnant women underwent a 75 g oral glucose tolerance test. Data on potential risk factors was collected and analysed using logistical regression analysis. Receiver operating characteristic (ROC) curves, sensitivity, specificity and predictive values were calculated for significant risk factors and a risk factor scoring variable was constructed.

Results

HIP was prevalent in 18.9% of the study population (16.3% GDM; 2.6% DIP). Increasing age and BMI as well as having a mother only or both parents with diabetes were significant independent risk factors for HIP. Among women attending the rural health centre a doubling of income corresponded to an 80% increased risk of HIP (OR 1.80, 95%CI 1.10–2.93; p = 0.019), whereas it was not significantly associated with HIP among women attending the other health centres. The performance of the individual risk factors and the constructed scoring variable differed substantially between the three health centres, but none of them were good enough to discriminate between those with and without HIP.

Conclusions

The findings highlight the importance of socio-economic circumstances and intergenerational risk transmission in the occurrence of HIP as well as the need for universal screening.     

Chiral separation of Frovatriptan isomers by HPLC using amylose based chiral stationary phase

A stereospecific HPLC method for separation of Frovatriptan enantiomers in bulk drug and pharmaceutical formulations was developed and validated on a normal-phase amylose derivertized chiral column. The effects of the organic modifiers namely 2-propanol, ethanol and diethyl amine (DEA) in the mobile phase were optimized to obtain the best enantiomeric separation. Calibration curves were linear over the range of 200-6150 ng/mL, with a regression coefficient (R(2)) of 0.9998. The limit of detection (LOD) and limit of quantification (LOQ) were 65 ng/mL and 200 ng/mL, respectively. The method was accurate and precise and suitable for the intended purpose. Analysis results were compared with the results obtained by using a validated chiral CE method and found to be in very good agreement. This method can be successfully applied to the enantiomeric purity analysis of Frovatriptan in pharmaceutical bulk drug samples and formulations.