Self-driving autonomous machines to engineer novel enzymes.

Can one design and automate a computational and experimental platform such that each platform iteratively guides and drives the other to achieve a pre-determined goal? Rapp and colleagues (2024) describe just this possibility in a paper that details a prototype of a self-driven laboratory that can navigate autonomously to yield an engineered enzyme with a desired attribute. This laboratory, rather, the automated protocol, is referred to by an acronym - SAMPLE. This refers to Self-driving Autonomous Machines for Protein Landscape Exploration. The paper describes a prototype involving the engineering of a glycoside hydrolase for enhanced thermostability. The 'brain', the computational component behind this automated system, was designed to learn protein sequence- function relationships from a curated dataset. These designer proteins were then evaluated by a fully automated robotic system that could synthesize and experimentally characterize the designed protein and provide feedback to the agent, i.e., the computational component, to fine-tune its understanding of the system. The SAMPLE agents were thus designed to continually refine their understanding of the protein landscape by actively acquiring information in the search process. As this intelligent agent learns protein sequence-function relationships from a curated, diverse dataset, this feedback is crucial to refine landscape exploration and the design of new proteins based on the updated hypothesis. In this prototype, four SAMPLE agents were tasked with this goal. The goal of each of these agents was to navigate the glycoside hydrolase landscape and identify enzymes with enhanced thermal tolerance. Differences in the search behavior of individual agents primarily arise from experimental measurement noise. However, despite differences in their search behavior, all four agents could converge on a thermostable glycoside hydrolase - a remarkable feat as it apparently did not need any human intervention.

Underlying factors determining grain morphologies in high-strength titanium alloys processed by additive manufacturing.

In recent research, additions of solute to Ti and some Ti-based alloys have been employed to produce equiaxed microstructures when processing these materials using additive manufacturing. The present study develops a computational scheme for guiding the selection of such alloying additions, and the minimum amounts required, to effect the columnar to equiaxed microstructural transition. We put forward two physical mechanisms that may produce this transition; the first and more commonly discussed is based on growth restriction factors, and the second on the increased freezing range effected by the alloying addition coupled with the imposed rapid cooling rates associated with AM techniques. We show in the research described here, involving a number of model binary as well as complex multi-component Ti alloys, and the use of two different AM approaches, that the latter mechanism is more reliable regarding prediction of the grain morphology resulting from given solute additions.

Optimizing image contrast of second phases in metal alloys.

Novel imaging strategies in the scanning electron microscope aimed at significantly improved image contrast of second phases in metal alloys are described. These include the use of low accelerating voltages, small working distances, and a novel detection system. Contrast is assessed as a function of voltage and optimized imaging conditions which result in much improved image quality are presented. These strategies are applied to two precipitation hardened Ni-base alloys, a cast single crystal and a hot isostatically pressed sample.

Relationship of Creatinine and Cystatin C-based Estimated Glomerular Filtration rates with Measured Glomerular Filtration Rate in Healthy Kidney Donors from South Asia.

Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) equation is currently recommended for the estimation of glomerular filtration rate (GFR). This retrospective study aimed to evaluate the correlation between creatinine and cysC-based estimated GFRs and measured GFR in healthy adults. Consecutive healthy adults who were accepted as voluntary kidney donors at our center between January 2008 and December 2012 were included in the study. The 336 individuals who comprised the study population had a mean age of 41.6 ± 11.8 years, male:female ratio 1:1.7, mean creatinine 0.9 ± 0.1 mg/dl, and mean cysC 0.8 ± 0.1 mg/dl. Mean measured GFR by Tc-99m diethylenetriaminepentaacetic acid using Gates method was 98.4 ± 21.2 ml/min/1.73 m2. The mean ± standard deviation of eGFRs by various formulae were as follows: Cockcroft-Gault (CG) = 88.1 ± 15.9 ml/min/1.73 m2, Modification of Diet in Renal Disease (MDRD) = 78 ± 14.7 ml/min/1.73 m2, CKD-EPI creatinine = 88.1 ± 15.5 ml/min/1.73 m2, CKD-EPI cysC = 97 ± 19.9 ml/min/1.73 m2, CKD-EPI creatinine-cysC (CKD-EPI cr-cysC) = 92.5 ± 14.1 ml/min/1.73 m2. The CKD-EPI cr-cysC equation had the highest accuracy, with 43% and 72% of values lying within ±10% and ±20% of the measured GFR, respectively. Bland-Altman analyses for levels of agreement showed least bias with CKD-EPI cysC overall and among females, while among males, CKD-EPI creatinine equation had the least bias. The CKD-EPI equation showed a higher performance than the MDRD and CG equation in GFR estimation of a healthy population. Among CKD-EPI equations, CKD-EPI cr-cysC had the highest accuracy and CKD-EPI cysC the least bias.

Hemostatic Abnormalities in Severe Renal Failure: Do They Bark or Bite?

Abnormal primary hemostasis is believed to be the most significant contributor to uremic bleeding. This study aimed to describe the prevalence and profile of primary and secondary hemostatic disorders in patients with chronic kidney disease (CKD) Stages 4 and 5 and to determine their association if any, with degree of uremia. Stages 4 and 5 predialysis CKD patients attending nephrology outpatient clinic were prospectively recruited and the following bleeding parameters were measured in all patients: platelet count, bleeding time (BT), Factor VIII assay, von Willebrand factor antigen (vWF:Ag), vWF:ristocetin cofactor activity (vWF:RCo), ratio of vWF:ristocetin cofactor activity to vWF antigen (vWF:RCo/vWF:Ag), prothrombin time (PT), and activated partial thromboplastin time (aPTT). Forty-five patients (80%, males) with a mean age of 39.4 years, 82% (n = 37) in Stage 5 CKD, were recruited for the study. The prevalence of thrombocytopenia was significantly higher among patients from West Bengal (15/26, 57.7%) compared to other study patients (2/19, 10.5%; P = 0.001); however, all had macrothrombocytes with normal BT, suggestive of the Harris syndrome. Factor VIII, vWF:Ag, vWF:RCo, vWF:RCo/vWF:Ag ratio, BT, PT, and aPTT were abnormal in 0 (0%), 0 (0%), 0 (0%), 4 (8.8%), 1 (2.2%), 7 (15.6%), and 5 (11.1%) patients, respectively. Except for thrombocytopenia, the prevalence of hemostatic abnormalities did not differ between CKD Stages 4 and 5. Hemostatic abnormalities are uncommon in Stages 4-5 CKD and except for thrombocytopenia, are not associated with degree of uremia. Constitutional macrothrombocytopenia is associated with normal BT even in CKD.

Production of Lipopeptide Biosurfactant by a Marine Nesterenkonia sp. and Its Application in Food Industry.

Biosurfactants are smart biomolecules which have wide spread application in medicines, processed foods, cosmetics as well as in bioremediation. In food industry, biosurfactants are used as emulsion stabilizing agents, antiadhesives, and antimicrobial/antibiofilm agents. Nowadays biosurfactant demands in industries has increased tremendously and therefore new bacterial strains are being explored for large scale production of biosurfactants. In this study, an actinobacterial strain MSA31 was isolated from a marine sponge Fasciospongia cavernosa which showed high activity in biosurfactant screening assays such as drop collapsing, oil displacement, lipase and emulsification. Lipopeptide produced by MSA31 was found to be thermostable which was evident in differential scanning calorimetry analysis. The spectral data obtained in the Fourier transform infrared spectroscopy showed the presence of aliphatic groups combined with peptide moiety which is a characteristic feature of lipopeptides. The stability index of lipopeptide MSA31 revealed "halo-alkali and thermal tolerant biosurfactant" which can be used in the food industry. Microtiter plate assay showed 125 µg/ml of lipopeptide was effective in reducing the biofilm formation activity of pathogenic multidrug resistant Staphylococcus aureus. The confocal laser scanning microscopic images provided further evidences that lipopeptide MSA31 was an effective antibiofilm agent. The antioxidant activity of lipopeptide MSA31 may be due to the presence of unsaturated fatty acid present in the molecule. The brine shrimp cytotoxicity assay showed lipopeptide MSA31 was non-toxic and can be used as food additives. Incorporation of lipopeptide MSA31 in muffin showed improved organoleptic qualities compared to positive and negative control. This study provides a valuable input for this lipopeptide to be used in food industry as an effective emulsifier, with good antioxidant activity and as a protective agent against S. aureus.

Determination of Redox Sensitivity in Structurally Similar Biological Redox Sensors.

Redox stimuli govern a variety of biological processes. The relative sensitivity of redox sensors plays an important role in providing a calibrated response to environmental stimuli and cellular homeostasis. This cellular machinery plays a crucial role in the human pathogen Mycobacterium tuberculosis as it encounters diverse microenvironments in the host. The redox sensory mechanism in M. tuberculosis is governed by two component and one-component systems, alongside a class of transcription factors called the extra cytoplasmic function (ECF) σ factors. ECF σ factors that govern the cellular response to redox stimuli are negatively regulated by forming a complex with proteins called zinc associated anti-σ factors (ZAS). ZAS proteins release their cognate σ factor in response to oxidative stress. The relative sensitivity of the ZAS sensors to redox processes dictate the concentration of free ECF σ factors in the cell. However, factors governing the redox threshold of these sensors remain unclear. We describe here, the molecular characterization of three σ factor/ZAS pairs-σL/RslA, σE/RseA, and σH/RshA-using a combination of biophysical and electrochemical techniques. This study reveals, conclusively, the differences in redox sensitivity in these proteins despite apparent structural similarity and rationalizes the hierarchy in the activation of the cognate ECF σ factors. Put together, the study provides a basis for examining sequence and conformational features that modulate redox sensitivity within the confines of a conserved structural scaffold. The findings provide the guiding principles for the design of intracellular redox sensors with tailored sensitivity and predictable redox thresholds, providing a much needed biochemical tool for understanding host-pathogen interaction.

Policies, Socioeconomic, Institutional and Biophysical Factors Influencing the Change from Rice to Sugarcane in Nong Bua Lamphu Province, Thailand.

For the past decade, Thailand pursued a strategy of promoting biofuel crop production, including sugarcane, to meet the ever increasing energy demand and to increase the income of farmers. This study analyzed the influence of policy instruments implemented to promote the sugarcane cultivation, farm household socioeconomic levels, biophysical, and institutional factors driving the conversion of rice fields into sugarcane farms. Primary information was collected from 230 farm households through a structured questionnaire. Relevant policy documents published by the responsible government agencies were the main sources of secondary information collected for policy analysis. The analyses revealed that farmers in the study area converted 25-75% of their rice fields into sugarcane farms as a result of implementation of policy instruments that made sugarcane financially far more attractive than rice. The results of the regression analysis showed that eight variables were found significant and positively influenced the conversion of rice fields into sugarcane farms. These were access to extension services, information, sugarcane loading stations, and ground water for irrigation, and duration of experience in sugarcane farming as well as household head's age and education. Irrespective of landholding size, the majority of farmers were not concerned about food security. In view of the finding, if feasible, in the form of additional price supports and commercial cane sugar index-based pricing strategies to promote sugarcane production should benefit all concerned farmers. Likewise, attention should be paid to deliver essential extension and marketing services in an effective way, particularly to those farmers who are not receiving such services.

Alterations in minute ventilation, maximum voluntary ventilation and dyspneic index in different trimesters of pregnancy.

Respiratory function in pregnancy is of special importance since the life of fetus depends primarily upon its oxygen supply. Thus this study was designed to evaluate the Minute ventilation (MV), Maximum Voluntary Ventilation (MVV) & Dyspneic Index (DI) in different trimesters of pregnancy & compare the results with non- pregnant control group. A cross-sectional study was carried out in 200 healthy women in the age range of 19-35 years with 50 subjects each in 1st, 2nd, 3rd trimesters of pregnancy and non-pregnant control group. We recorded respiratory parameters in study and control groups. Statistical analysis was done by ANOVA and Tukey Krammer post Hoc tests. It was observed that there was a significant decrease in MVV and dyspneic index in all trimesters of pregnancy and an insignificant variation in MV when compared to the control group. The changes in pulmonary function are influenced by the mechanical pressure of enlarging gravid uterus, elevating the diaphragm and restricting the movements of lungs thus hampering forceful expiration. The decrease seen in MVV in 1st trimester might be due to bronchoconstriction effect of decreased alveolar Pco2 on the bronchial smooth muscles.

Effect of yoga on oxidative stress in elderly with grade-I hypertension: a randomized controlled study.

BACKGROUND AND OBJECTIVES: Hypertension, especially in elderly is a strong risk factor for cardiovascular mortality and morbidity. Oxidative stress has been implicated as one of the underlying cause of hypertension. Yoga has been found to control hypertension in the elderly, but the underlying benefits of mechanism in relation to oxidative stress regulation remains unclear. The purpose of the study was to investigate the effect of yoga on oxidative stress in elderly with Grade-I hypertension. METHODS: An open parallel-arm randomised controlled study was conducted at BLDE University's Shri B.M.Patil Medical College, Hospital and Research Centre, India on elderly male individuals with Grade-I hypertension (n=57, age 60-80 years). Study (Yoga) group was assigned for yoga intervention and control group for walking for one hour in the morning for six days in a week for three months under the supervision of yoga instructor and physical training instructor respectively. Serum malondialdehyde (MDA) as an indicator of oxidative stress and antioxidants such as serum superoxide dismutase (SOD), reduced glutathione (GSH) and vitamin C levels were estimated. RESULTS: Yoga practice for three months has significantly reduced serum MDA level (p<0.001), and enhanced antioxidants level such as SOD activity (p=0.007), serum GSH (p=0.002) and vitamin C (p=0.002). In the control group, we observed a significant increase in serum MDA level (p=0.04) and reduction in serum vitamin C level (p=0.015) with no significant difference in the SOD activity and GSH level. CONCLUSION: These findings suggest that yoga is an effective means to reduce oxidative stress and to improve antioxidant defense in elderly hypertensive individuals.

Structural basis for the redox sensitivity of the Mycobacterium tuberculosis SigK-RskA σ-anti-σ complex.

The host-pathogen interactions in Mycobacterium tuberculosis infection are significantly influenced by redox stimuli and alterations in the levels of secreted antigens. The extracytoplasmic function (ECF) σ factor σ(K) governs the transcription of the serodominant antigens MPT70 and MPT83. The cellular levels of σ(K) are regulated by the membrane-associated anti-σ(K) (RskA) that localizes σ(K) in an inactive complex. The crystal structure of M. tuberculosis σ(K) in complex with the cytosolic domain of RskA (RskAcyto) revealed a disulfide bridge in the -35 promoter-interaction region of σ(K). Biochemical experiments reveal that the redox potential of the disulfide-forming cysteines in σ(K) is consistent with its role as a sensor. The disulfide bond in σ(K) influences the stability of the σ(K)-RskAcyto complex but does not interfere with σ(K)-promoter DNA interactions. It is noted that these disulfide-forming cysteines are conserved across homologues, suggesting that this could be a general mechanism for redox-sensitive transcription regulation.

Titania nanotube-modified screen printed carbon electrodes enhance the sensitivity in the electrochemical detection of proteins.

The use of titania nanotubes (TiO2-NT) as the working electrode provides a substantial improvement in the electrochemical detection of proteins. A biosensor designed using this strategy provided a robust method to detect protein samples at very low concentrations (Cprotein ca 1ng/µl). Reproducible measurements on protein samples at this concentration (Ip,a of 80+1.2µA) could be achieved using a sample volume of ca 30µl. We demonstrate the feasibility of this strategy for the accurate detection of penicillin binding protein, PBP2a, a marker for methicillin resistant Staphylococcus aureus (MRSA). The selectivity and efficiency of this sensor were also validated using other diverse protein preparations such as a recombinant protein tyrosine phosphatase (PTP10D) and bovine serum albumin (BSA). This electrochemical method also presents a substantial improvement in the time taken (few minutes) when compared to conventional enzyme-linked immunosorbent assay (ELISA) protocols. It is envisaged that this sensor could substantially aid in the rapid diagnosis of bacterial infections in resource strapped environments.

Eight-and-a-half syndrome: a rare presentation of pontine infarction.

"Eight-and-a-half" syndrome is "one-and-a-half" syndrome (conjugated horizontal gaze palsy and internuclear ophthalmoplegia) plus ipsilateral fascicular cranial nerve seventh palsy. This rare condition, particularly when isolated, is caused by circumscribed lesions of the pontine tegmentum involving the abducens nucleus, the ipsilateral medial longitudinal fasciculus, and the adjacent facial colliculus. Its recognition is therefore of considerable diagnostic value. We report a case of a 65-year-old man who presented with eight-and-a-half syndrome in which brain magnetic resonance imaging scan revealed a lacunar pontine infarction.

Nature of the interfaces between the constituent phases in the high entropy alloy CoCrCuFeNiAl.

The interfaces between the phase separated regions in the dendritic grains of laser-deposited samples of the high entropy alloy CoCrCuFeNiAl have been studied using aberration-corrected analytical (scanning) transmission electron microscopy ((S)TEM). The compositional variations have been determined using energy dispersive x-ray spectroscopy (EDS) in (S)TEM. It was found that between B2, consisting mainly of Al, Ni, Co, and Fe, and disordered bcc phase, consisting mainly of Cr and Fe, there is a transition region, approximately 1.5 nm in width, over which the chemical composition changes from the B2 to that of the bcc phase. The crystal structure of this interfacial region is also B2, but with very different sublattice occupancy than that of the adjacent B2 compound. The structural aspects of the interface between the ordered B2 phase and the disordered bcc phase have been characterized using high angle annular dark-field (HAADF) imaging in STEM. It has been determined that the interfaces are essentially coherent, with the lattice parameters of the two B2 regions and the disordered bcc phase being more or less the same, the uncertainty arising from possible relaxations from the proximity of the surfaces of the thin foils used in imaging of the microstructures. Direct observations show that there is a planar continuity between all three constituent phases.

Structural insights into the role of Bacillus subtilis YwfH (BacG) in tetrahydrotyrosine synthesis.

The synthesis of the dipeptide antibiotic bacilysin involves the sequential action of multiple enzymes in the bac operon. YwfH (also referred to as BacG) catalyzes the stereoselective reduction of dihydro-hydroxyphenylpyruvate (H2HPP) to tetrahydro-hydroxyphenylpyruvate (H4HPP) in this biosynthetic pathway. YwfH is an NADPH-dependent reductase that facilitates the conjugate addition of a hydride at the C4 olefin terminus of H2HPP. Here, the structure of YwfH is described at three conformational steps: the apo form, an apo-like conformation and the NADPH complex. YwfH is structurally similar to other characterized short-chain dehydrogenase/reductases despite having marginal sequence similarity. The structures of YwfH in different conformational states provide a rationale for the ping-pong reaction mechanism. The identification and role of the residues in the catalytic tetrad (Lys113-Tyr117-Ser155-Asn158) in proton transfer were examined by mutational analysis. Together, the structures and biochemical features revealed synchronized conformational changes that facilitate cofactor specificity and catalysis of H4HPP formation en route to tetrahydrotyrosine synthesis.

Structure of an amidohydrolase, SACOL0085, from methicillin-resistant Staphylococcus aureus COL.

Staphylococcus aureus is an opportunistic pathogen that rapidly acquires resistance to frontline antibiotics. The characterization of novel protein targets from this bacterium is thus an important step towards future therapeutic strategies. Here, the crystal structure of an amidohydrolase, SACOL0085, from S. aureus COL is described. SACOL0085 is a member of the M20D family of peptidases. Unlike other M20D peptidases, which are either monomers or dimers, SACOL0085 adopts a butterfly-shaped homotetrameric arrangement with extensive intersubunit interactions. Each subunit of SACOL0085 contains two Mn(2+) ions at the active site. A conserved cysteine residue at the active site distinguishes M20D peptidases from other M20 family members. This cysteine, Cys103, serves as bidentate ligand coordinating both Mn(2+) ions in SACOL0085.

Effect of yoga on short-term heart rate variability measure as a stress index in subjunior cyclists: a pilot study.

Subjunior athletes experience mental stress due to pressure from the coach, teachers and parents for better performance. Stress, if remains for longer period and not managed appropriately can leads to negative physical, mental and cognitive impact on children. The present study was aimed to evaluate the effect of integrated yoga module on heart rate variability (HRV) measure as a stress index in subjunior cyclists. Fast furrier transform technique of frequency domain method was used for the analysis of HRV. We have found a significant increase in high frequency (HF) component by 14.64% (P < 0.05) and decrease in the low frequency component (LF) of HRV spectrum by 5.52% (P < 0.05) and a decrease in LF/HF ratio by 19.63% (P < 0.01) in yoga group. In the control group, there was decrease in the HF component and, no significant difference in the LF component of HRV spectrum and LF/HF ratio. The results show that yoga practice decreases sympathetic activity and causes a shift in the autonomic balance towards parasympathetic dominance indicating a reduction in stress. In conclusion, yoga practice helps to reduce stress by optimizing the autonomic functions. So, it is suggested to incorporate yoga module as a regular feature to keep subjunior athletes both mentally and physically fit.

Biodiversity monitoring at the Tonle Sap Lake of Cambodia: a comparative assessment of local methods.

This paper assesses local biodiversity monitoring methods practiced in the Tonle Sap Lake of Cambodia. For the assessment we used the following criteria: methodological rigor, perceived cost, ease of use (user friendliness), compatibility with existing activities, and effectiveness of intervention. Constraints and opportunities for execution of the methods were also considered. Information was collected by use of: (1) key informant interview, (2) focus group discussion, and (3) researcher's observation. The monitoring methods for fish, birds, reptiles, mammals and vegetation practiced in the research area have their unique characteristics of generating data on biodiversity and biological resources. Most of the methods, however, serve the purpose of monitoring biological resources rather than biodiversity. There is potential that the information gained through local monitoring methods can provide input for long-term management and strategic planning. In order to realize this potential, the local monitoring methods should be better integrated with each other, adjusted to existing norms and regulations, and institutionalized within community-based organization structures.

Inter-domain interactions influence the stability and catalytic activity of the bi-domain protein tyrosine phosphatase PTP99A.

The two protein tyrosine phosphatase (PTP) domains in bi-domain PTPs share high sequence and structural similarity. However, only one of the two PTP domains is catalytically active. Here we describe biochemical studies on the two tandem PTP domains of the bi-domain PTP, PTP99A. Phosphatase activity, monitored using small molecule as well as peptide substrates, revealed that the inactive (D2) domain activates the catalytic (D1) domain. Thermodynamic measurements suggest that the inactive D2 domain stabilizes the bi-domain (D1-D2) protein. The mechanism by which the D2 domain activates and stabilizes the bi-domain protein is governed by few interactions at the inter-domain interface. In particular, mutating Lys990 at the interface attenuates inter-domain communication. This residue is located at a structurally equivalent location to the so-called allosteric site of the canonical single domain PTP, PTP1B. These observations suggest functional optimization in bi-domain PTPs whereby the inactive PTP domain modulates the catalytic activity of the bi-domain enzyme.

Role of exercise and nutrition on cardiopulmonary fitness and pulmonary functions on residential and non-residential school children.

Physical fitness is the prime criterion for survival and to lead a healthy life. Our aim is to find out effect of exercise and nutrition on physical fitness on growing children with scientific records. The present study was designed on healthy school children of a Residential-Sainik (100) and Non-Residential (100) school children (12-16 yrs) of Bijapur. To evaluate cardiopulmonary fitness parameters included are VO2Max (ml/kg/min) and Physical Fitness Index (PFI %). Harvard Step Test determined VO2 Max and PFI. Also recorded pulmonary function parameters like Forced Expiratory Volume in 1 sec (FEV1 in %) by recording spirometry. Peak Expiratory Flow Rate (PEFR in L/Min) by Peak flow meter and Maximal Expiratory Pressure (MEP in mmHg) by modified Black's apparatus. We found statistically significant higher values (p = 0.000) of VO2Max, PFI, FEV1, PEFR and MEP in residential school children compared to nonresidential school children higher. So, our study shows that regular exercise and nutritious food increase the cardiopulmonary fitness values and pulmonary functions in Residential school children.