Isolation and Characterization of a Nitrile-Hydrolysing Bacterium Isoptericola variabilis RGT01.

A nitrile-hydrolysing bacterium, identified as Isoptericola variabilis RGT01, was isolated from industrial effluent through enrichment culture technique using acrylonitrile as the carbon source. Whole cells of this microorganism exhibited a broad range of nitrile-hydrolysing activity as they hydrolysed five aliphatic nitriles (acetonitrile, acrylonitrile, propionitrile, butyronitrile and valeronitrile), two aromatic nitriles (benzonitrile and m-Tolunitrile) and two arylacetonitriles (4-Methoxyphenyl acetonitrile and phenoxyacetonitrile). The nitrile-hydrolysing activity was inducible in nature and acetonitrile proved to be the most efficient inducer. Minimal salt medium supplemented with 50 mM acetonitrile, an incubation temperature of 30 °C with 2 % v/v inoculum, at 200 rpm and incubation of 48 h were found to be the optimal conditions for maximum production (2.64 ± 0.12 U/mg) of nitrile-hydrolysing activity. This activity was stable at 30 °C as it retained around 86 % activity after 4 h at this temperature, but was thermolabile with a half-life of 120 min and 45 min at 40 °C and 50 °C respectively.

Laboratory evolution of enantiocomplementary Candida antarctica lipase B mutants with broad substrate scope.

Candida antarctica lipase B (CALB) is a robust and easily expressed enzyme used widely in academic and industrial laboratories with many different kinds of applications. In fine chemicals production, examples include acylating kinetic resolution of racemic secondary alcohols and amines as well as desymmetrization of prochiral diols (or the reverse hydrolytic reactions). However, in the case of hydrolytic kinetic resolution of esters or esterifying kinetic resolution of acids in which chirality resides in the carboxylic acid part of the substrate, rate and stereoselectivity are generally poor. In the present study, directed evolution based on iterative saturation mutagenesis was applied to solve the latter problem. Mutants with highly improved activity and enantioselectivity relative to wild-type CALB were evolved for the hydrolytic kinetic resolution of p-nitrophenyl 2-phenylpropanoate, with the selectivity factor increasing from E = 1.2 (S) to E = 72 (S) or reverting to E = 42 (R) on an optional basis. Surprisingly, point mutations both in the acyl and alcohol pockets of CALB proved to be necessary. Some of the evolved CALB mutants are also efficient biocatalysts in the kinetic resolution of other chiral esters without performing new mutagenesis experiments. Another noteworthy result concerns the finding that enantiocomplementary CALB mutants for α-substituted carboxylic acid esters also show stereocomplementarity in the hydrolytic kinetic resolution of esters derived from chiral secondary alcohols. Insight into the source of stereoselectivity was gained by molecular dynamics simulations and docking experiments.

How Important is the Tunnel Position in Outcomes Post-ACL Reconstruction: A 3D CT-Based Study.

BACKGROUND: Drilling the femoral and tibial tunnels at their anatomical locations are critical for good outcomes and involve seeing the footprints well. We intended to compare two techniques of drilling the tunnels and the patient-reported outcomes and knee stability of patients undergoing single bundle ACL reconstruction using 3D CT to evaluate if the tunnels were anatomical or not. MATERIALS AND METHODS: Sixty single bundle ACL reconstructions were analyzed, 30 each with Technique A and B. Pre-operative and after a minimum 27 month follow-up Lysholm, IKDC, Tegner score, hop test, and Lachman test were noted. 3D CT was done to classify femoral tunnels positions as being well placed, slightly or grossly misplaced and tibial tunnels as optimal or suboptimal and compared. RESULTS: Sixty ACL reconstructions had full follow-up with a mean follow-up of 34 months. There was no significant difference between tunnel positions between the two techniques. Well-placed femoral tunnel had better Lysholm score (62.2 ± 16.2 v/s 48.5 ± 17.2, p 0.002) and IKDC score (62.5 ± 14.3 v/s 52.7 ± 15.1, p 0.012).). Those who had their surgeries within 3 months of their injury had better hop test (4.4 ± 0.9 v/s 3.9 ± 1, p 0.034) and IKDC scores (62.5 ± 15.8 v/s 33.2 ± 13.8, p 0.026) as compared to those that had surgery done after 3 months. CONCLUSION: Tibial tunnel positions were optimal in most cases and did not differ between the two techniques. Well-placed femoral tunnels and surgeries done within 3 months of the injury produced best results.

Secondary infections modify the overall course of hospitalized patients with COVID-19: a retrospective study from a network of hospitals across North India.

Objective: To gain better insight into the extent of secondary bacterial and fungal infections in hospitalized patients in India, and to assess how these alter the course of coronavirus disease 2019 (COVID-19) so that control measures can be suggested. Methods: In this retrospective, multicentre study, the data of all patients who tested positive for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) on reverse transcriptase polymerase chain reaction (RT-PCR), admitted to hospital between March 2020 and July 2021, were accessed from the electronic health records of a network of 10 hospitals across five states in North India. Results: Of 19,852 patients testing positive for SARS-CoV-2 on RT-PCR and admitted to the study hospitals during the study period, 1940 (9.8%) patients developed secondary infections (SIs). Patients with SIs were, on average, 8 years older than patients without SIs (median age 62.6 vs 54.3 years; P<0.001). The risk of SIs was significantly (P<0.001) associated with age, severity of disease at admission, diabetes, admission to the intensive care unit (ICU), and ventilator use. The most common site of infection was urine (41.7%), followed by blood (30.8%) and sputum/bronchoalveolar lavage/endotracheal fluid (24.8%); the least common was pus/wound discharge (2.6%). Gram-negative bacilli (GNB) were the most common organisms (63.2%), followed by Gram-positive cocci (GPC) (19.6%) and fungi (17.3%). Most patients with SIs were on multiple antimicrobials. The most commonly used antibiotics against GNB were beta-lactam/beta-lactamase inhibitors (76.9%), carbapenems (57.7%), cephalosporins (53.9%), and antibiotics against carbapenem-resistant Enterobacteriaceae (47.1%). Empirical use of antibiotics against GPC was seen in 58.9% of patients with SIs, and empirical use of antifungals was observed in 56.9% of patients with SIs. The average length of hospital stay for patients with SIs was almost twice as long as that of patients without SIs (median 13 vs 7 days). Overall mortality among patients with SIs (40.3%) was more than eight times higher than that among patients without SIs (4.6%). Only 1.2% of patients with SIs with mild COVID-19 at admission died, compared with 17.5% of those with moderate COVID-19 at admission and 58.5% of those with severe COVID-19 at admission (P<0.001). The mortality rate was highest in patients with bloodstream infections (49.8%), followed by those with hospital-acquired pneumonia (47.9%), urinary tract infections (29.4%), and skin and soft tissue infections (29.4%). The mortality rate in patients with diabetes with SIs was 45.2%, compared with 34.3% in those without diabetes (P<0.001). Conclusions: SIs complicate the course of patients hospitalized with COVID-19. These patients tend to have a much longer hospital stay, a higher requirement for oxygen and ICU care, and a significantly higher mortality rate compared with those without SIs. The groups most vulnerable to SIs are patients with more severe COVID-19, elderly patients and patients with diabetes. Judicious empirical use of combination antimicrobials in these groups of vulnerable patients can save lives. It is desirable to have region- or country-specific guidelines for appropriate use of antibiotics and antifungals to prevent their overuse.

Does Viewing the ACL Femoral Footprint End on Using a High Medial Portal Produce Better Tunnel Placement as Compared to Viewing it from a Lateral Portal while Drilling: A 3D CT-based Pilot Study.

BACKGROUND: Anatomical placement of an ACL graft is critical to the clinical outcome of an ACL reconstruction. The purpose of the study was to compare the conventional technique of drilling a femoral tunnel from a medial portal while viewing it from a lateral portal versus viewing it from a high anteromedial portal and drilling it from a low medial portal. We hypothesized that the high anteromedial portal possibly provides an end-on view of the tunnel and would lead to a more favourable tunnel position. MATERIALS AND METHODS: Sixty patients underwent arthroscopic single-bundle ACL reconstruction with a soft tissue graft. All patients had the same surgeon and same surgical technique (except viewing portal). Each patient underwent a 3D-CT 1-week post-op. We classified the patients into two groups (technique A and B) of 30 each. Postoperative tunnel position in each patient was visualized on CT scan with 3D reconstruction by the quadrant method described by Bernard et al. The distance of the centre of the femoral tunnel from the superior and posterior border of the Bernard frame was obtained in each patient of the two groups and mean value in each group was obtained and compared by Student 't' test at 5% level of significance. The position of the femoral tunnels was also studied in reference to the lateral intercondylar ridge and classified into type I (well placed), type II (moderately malpositioned) and type III (grossly malpositioned). RESULTS: The mean distances in technique A from the posterior and superior border of the lateral femoral condyle was 35.8 ± 8.2 and 35.2 ± 6.7, and in technique B was 38.1 ± 8.6 and 35.8 ± 9.4. The two groups did not differ significantly (P > 0.05) in femoral tunnel position as visualized on 3D-CT scan postoperatively. 53.3% patients had well-placed tunnels (type I) overall with 56.67% of technique A and 50% of technique B in this group. None of the tunnels were grossly malpositioned. CONCLUSIONS: The absence of any statistically significant difference between the groups suggests that a high anterolateral portal close to the patellar tendon gives an almost complete view of the lateral aspect of the notch, if not end on. Both techniques produced a majority of well placed (type I) tunnels.

Knowledge-guided laboratory evolution of protein thermolability.

In rare but nevertheless important cases it is of practical interest to decrease the thermostability of an enzyme, that is, to increase thermolability in a controlled manner. In the present model study, this unconventional goal has been reached by applying directed evolution to the lipase from Pseudomonas aeruginosa (PAL). By utilizing the B-factor iterative test (B-FIT), previously developed to increase the thermostability of enzymes, it was possible to reduce the T(15)(50) value from 71.6 degrees C in the case of wild type (WT-PAL) to 35.6 degrees C (best mutant) without affecting the catalytic profile in terms of substrate acceptance or enantioselectivity at room temperature. Accordingly, saturation mutagenesis was performed at sites in PAL, which on the basis of its X-ray structure, have the lowest B-factors indicative of high rigidity. Focused mutations were introduced which can be expected to decrease rigidity, the ensuing increased flexibility leading to higher thermolability without changing the actual catalytic profile.

A genetic selection system for evolving enantioselectivity of enzymes.

As an alternative to screening in the directed evolution of enantioselective enzymes, a selection system has been implemented for a lipase-catalyzed hydrolytic kinetic resolution of a chiral ester.

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates.

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

Development, characterization and application of a new epithelial cell line from caudal fin of Pangasianodon hypophthalmus (Sauvage 1878).

A cell line, designated as PHF, has been established from caudal fin of Pangasianodon hypophthalmus. The cell line was developed using explant method and PHF cells have been subcultured for more than 72 passages over a period of 14 months. The cells were able to grow at temperatures between 24 and 32°â€¯C, with an optimum temperature of 28°â€¯C. The growth rate of PHF cells was directly proportional to FBS concentration, with optimum growth observed at 20% FBS concentration. On the basis of immunophenotyping assay, PHF cells were confirmed to be of epithelial type. Karyotyping of PHF cells revealed diploid number of chromosomes (2n = 60) at 39th and 65th passage, which indicated that the developed cell line is chromosomally stable. The origin of the cell line was confirmed by amplification and sequencing of cytochrome oxidase c subunit I and 16S rRNA genes. The cell line was tested for Mycoplasma contamination and found to be negative. The cells were successfully transfected with GFP reporter gene suggesting that the developed cell line could be utilized for gene expression studies in future. The cell line could be successfully employed for evaluating the cytotoxicity of heavy metals, namely mercuric chloride and sodium arsenite suggesting that PHF cell line can be potential surrogate for whole fish for studying the cytotoxicity of water soluble compounds. The result of virus susceptibility to tilapia lake virus (TiLV) revealed that PHF cells were refractory to TiLV virus. The newly established cell line would be a useful tool for investigating disease outbreaks particularly of viral etiology, transgenic as well as cytotoxicity studies.

Response surface optimization of the critical medium components for carbonyl reductase production by Candida viswanathii MTCC 5158.

Culture conditions were optimized for the growth and carbonyl reductase production by a novel yeast strain Candida viswanathii. Response surface methodology was applied for the critical medium components (initial pH, mannitol, yeast extract and calcium chloride) identified earlier by one-factor-at-a-time approach. Central composite design was used for the optimization studies. Using this methodology, the optimal values for the concentration of mannitol, initial pH, yeast extract and calcium chloride were 1.9, 7.5, 1.6 and 4, respectively. This medium was projected to produce, theoretically, growth having an optical density of 1.1 (600 nm) and an enzyme activity of 81.5 U/ml. Using this optimized medium, an experimental growth of 1.1 OD (600 nm) and enzyme activity 80.9 U/ml verified the applied methodology. This approach for medium optimization led to an enhancement of the growth and enzyme activity by 1.3 and 2.3 times higher, respectively, as compared to the unoptimized media.

Increasing the stability of an enzyme toward hostile organic solvents by directed evolution based on iterative saturation mutagenesis using the B-FIT method.

Mutants of the lipase from Bacillus subtilis, previously engineered for enhanced thermostability using directed evolution based on the B-FIT method, show significantly increased tolerance to hostile organic solvents.

Optimization of process parameters for the production of carbonyl reductase by Candida viswanathii in a laboratory-scale fermentor.

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg(-1)) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25 degrees C. The enzyme activity was twice as high (56 Umg(-1)) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.

Enantioselective reduction of acetophenone and its derivatives with a new yeast isolate Candida tropicalis PBR-2 MTCC 5158.

The enantioselective bioreduction of acetophenone and its various analogues has been carried out using a new yeast strain, Candida tropicalis MTCC 5158, to obtain the corresponding (S)-aryl ethanols with good yield and almost absolute enantioselectivity. The catalytic ability of this microbial strain for acetophenone reduction has been examined and also various parameters of the bioreduction reaction have been optimized. Studies on the catalytic performance showed that this microorganism is capable of carrying out the reduction in a broad range of pH (3-10) and temperature (25-40 degrees C), making it a more versatile biocatalyst. The preparative scale bioreduction of acetophenone using resting cells of Candida tropicalis yielded S-(-)-1-phenyl ethanol with 43% yield and >99% enantiomeric excess.

Optimization of physicochemical parameters for the enhancement of carbonyl reductase production by Candida viswanathii.

Culture conditions have been optimized for a newly isolated yeast strain Candida viswanathii PBR2 which is capable of reducing a wide variety of aryl ketones with high stereospecificity. Studies on the culture conditions and catalytic performance of this microorganism showed that the carbonyl reductase occurs constitutively in the cells and its production is enhanced by feeding with acetophenone (2 mM) during the early period of cultivation. Mannitol (1%, wv(-1)) was found to be beneficial both for growth and enzyme production. Supplementation of the media with yeast extract (1.0%, wv(-1)) and Ca(2+ ) (4 mM) enhanced the enzyme production. The optimal temperature and pH for the growth and enzyme production were 25 degrees C and 9.0, respectively. Excellent conversions along with almost absolute enantioselectivity were observed when the resting cells of this yeast strain were exploited to carry out the stereoselective reduction of a number of aryl ketones.

Biotransformations for the production of the chiral drug (S)-Duloxetine catalyzed by a novel isolate of Candida tropicalis.

A yeast strain, Candida tropicalis PBR-2, isolated from soil, is capable of carrying out the enantioselective reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine to (S)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine, a key intermediate in the synthesis of the chiral drug (S)-Duloxetine. The organism produced the enantiopure (S)-alcohol with a good yield (> 80%) and almost absolute enantioselectivity, with an enantiomeric excess (ee) > 99%. Parameters of the bioreduction reaction were optimized and the optimal temperature and pH for the reduction were found to be 30 degrees C and 7.0, respectively. The optimized substrate and the resting cell concentration were 1 g/l and 250 g/l, respectively. The preparative-scale reaction using resting cells of C. tropicalis yielded the (S)-alcohol at 84-88% conversion and ee > 99%.

High-performance liquid chromatographic method for the simultaneous estimation of the key intermediates of duloxetine.

A simple reversed-phase high-performance liquid chromatographic method employing C-18 column has been developed for simultaneous analysis of three intermediates in the synthesis of S-duloxetine, the antidepressant drug, viz., 2-acetyl thiophene (AT), N,N-dimethyl-3-keto-(2-thienyl)-propanamine (DKTP) and (S)-N,N-dimethyl-3-hydroxy-(2-thienyl)-propanamine (DHTP). Good separations were achieved by employing an isocratic system using acetonitrile and 0.05M phosphate buffer (pH 7.0) containing 0.02% diethylamine. The detection was carried out at 241nm. The method was validated for linearity, range, accuracy and precision. The developed method was applied for monitoring the progress of chemical synthesis of DKTP from AT followed by the biocatalytic reduction of DKTP to DHTP as the disappearance of the substrate and formation of the product can be monitored simultaneously by the present method.