Computational approaches for automatic structural analysis of large biomolecular complexes

We present computational solutions to two problemsof macromolecular structure interpretation from reconstructedthree-dimensional electron microscopy (3D-EM) maps of largebio-molecular complexes at intermediate resolution (5A-15A). Thetwo problems addressed are: (a) 3D structural alignment (matching)between identified and segmented 3D maps of structure units(e.g. trimeric configuration of proteins), and (b) the secondarystructure identification of a segmented protein 3D map (i.e.locations of a-helices, b -sheets). For problem (a), we presentan efficient algorithm to correlate spatially (and structurally)two 3D maps of structure units. Besides providing a similarityscore between structure units, the algorithm yields an effectivetechnique for resolution refinement of repeated structure units,by 3D alignment and averaging. For problem (b), we present anefficient algorithm to compute eigenvalues and link eigenvectorsof a Gaussian convoluted structure tensor derived from theprotein 3D Map, thereby identifying and locating secondarystructural motifs of proteins. The efficiency and performanceof our approach is demonstrated on several experimentallyreconstructed 3D maps of virus capsid shells from single-particlecryo-EM, as well as computationally simulated protein structuredensity 3D maps generated from protein model entries in theProtein Data Bank.     

What should prompt an urgent referral to a community mental health team?

Background There is often little guidance to advise general practitioners on whether a referral to a community mental health team should be classified as 'urgent' or not.Aims (1) To identify the proportion and appropriateness of referrals considered urgent by the referrer; (2) To develop a set of criteria to guide what should constitute an 'urgent' referral.Methods One hundred consecutive referral letters to a community mental health team were analysed to determine the proportion that were considered urgent by the referrer compared to a consensus panel of psychiatrists. A Delphi group was then used to develop a set of criteria to guide referrers as to what should be regarded as an urgent referral.Results Thirty-three percent of referrals were deemed urgent by the referrer, compared to 17% by the psychiatric consensus panel, with little agreement between the two (kappa = 0.021, P = 0.013). Referrals that were made using a single assessment process (SAP) form were significantly more likely to be inappropriately marked as being urgent (P < 0.001). A set of 12 criteria was developed using the Delphi technique.Conclusions There was significant disagreement between the referrers and the assessing team as to which referrals required urgent attention. The findings justified the creation of guidelines, and this paper outlines a set of 12 criteria to guide what should prompt an urgent referral.     

Simultaneous Biodegradation of Polyaromatic Hydrocarbons by a <Emphasis Type="Italic">Stenotrophomonas</Emphasis> sp: Characterization of <Emphasis Type="Italic">nid</Emphasis> Genes and Effect of Surfactants on Degradation

A polyaromatic hydrocarbon degrading bacterium was isolated from a petroleum contaminated site and designated as Stenotrophomonas sp. strain IITR87. It was found to utilize pyrene, phenanthrene and benzo(a)pyrene as sole carbon source, but not anthracene, chrysene and fluoranthene. Gas chromatography and mass spectroscopy analysis resulted in identification of pyrene metabolites namely monohydroxypyrene, 4-oxa-pyrene-5-one, dimethoxypyrene and monohydroxyphenanthrene. Southern hybridization using naphthalene dioxygenase gene (nidA) as probe against the DNA of strain IITR87 revealed the presence of nidA gene. PCR analysis suggests dispersed occurrence of nid genes in the genome instead of a cluster as reported in a PAH-degrading Mycobacterium vanbaalenii PYR-1. The nid genes in strain IITR87, dioxygenase large subunit (nidA), naphthalene dioxygenase small subunit (nidB) and aldehyde dehydrogenase gene (nidD) showed more than 97 % identity to the reported nid genes from Mycobacterium vanbaalenii PYR-1. Most significantly, the biodegradation of PAHs was enhanced 25–60 % in the presence of surfactants rhamnolipid and Triton X-100 due to increased solubilization and bioavailability. These results could be useful for the improved biodegradation of high-molecular-weight PAHs in contaminated habitats.     

Nurturing diversity and inclusion in AI in Biomedicine through a virtual summer program for high school students

Artificial Intelligence (AI) has the power to improve our lives through a wide variety of applications, many of which fall into the healthcare space; however, a lack of diversity is contributing to limitations in how broadly AI can help people. The UCSF AI4ALL program was established in 2019 to address this issue by targeting high school students from underrepresented backgrounds in AI, giving them a chance to learn about AI with a focus on biomedicine, and promoting diversity and inclusion. In 2020, the UCSF AI4ALL three-week program was held entirely online due to the COVID-19 pandemic. Thus, students participated virtually to gain experience with AI, interact with diverse role models in AI, and learn about advancing health through AI. Specifically, they attended lectures in coding and AI, received an in-depth research experience through hands-on projects exploring COVID-19, and engaged in mentoring and personal development sessions with faculty, researchers, industry professionals, and undergraduate and graduate students, many of whom were women and from underrepresented racial and ethnic backgrounds. At the conclusion of the program, the students presented the results of their research projects at the final symposium. Comparison of pre- and post-program survey responses from students demonstrated that after the program, significantly more students were familiar with how to work with data and to evaluate and apply machine learning algorithms. There were also nominally significant increases in the students’ knowing people in AI from historically underrepresented groups, feeling confident in discussing AI, and being aware of careers in AI. We found that we were able to engage young students in AI via our online training program and nurture greater diversity in AI. This work can guide AI training programs aspiring to engage and educate students entirely online, and motivate people in AI to strive towards increasing diversity and inclusion in this field.     

A procedure for isolation of human protein C and protein S as by-products of the purification of factors VII, IX, X and prothrombin

A DEAE-Sephadex column chromatography step utilized to purify human Factor VII consistently yields a protein peak between the factor VII activity peak and prothrombin, factor X and factor IX activity peak (S.P. Bajaj, S.I. Rapaport, and S.F. Brown: J. Biol. Chem. 251, 253-259, 1981). We now report that this protein peak contains protein C and protein S. Preparative disc polyacrylamide gel electrophoresis of the proteins in this peak permitted a complete separation of protein C from protein S. Protein C at this step usually contained approximately 5-10% of Factor X, which could be removed by a goat anti-human Factor X antibody column. For a typical preparation, starting with 10L of plasma, the yield of Protein C was 5 mg and of protein S was 4 mg. Both proteins revealed apparent homogeneity in sodium dodecyl sulfate gel electrophoretic system. beta-Protein C and beta-protein S were not observed in our preparations starting with plasma collected directly into citrate anticoagulant containing benzamidine and soybean trypsin inhibitor, suggesting that these beta forms of protein C and protein S, isolated by other investigators, are slightly degraded forms of the native proteins. Antisera generated to these proteins were monospecific and could be used to monitor column fractions during purification. When examined by immunoelectrophoresis, the electrophoretic mobility of protein S in plasma was slower than that of isolated protein S. When exposed to plasmin, protein C was activated slightly and then rapidly degraded.     

Experimental and theoretical evidence supporting the role of Gly363 in blood coagulation factor IXa (Gly193 in chymotrypsin) for proper activation of the proenzyme

Factor IX is the zymogen of the serine protease factor IXa involved in blood coagulation. In addition to a catalytic domain homologous to the chymotrypsin family, it has Ca2+, phospholipid, and factor VIIIa binding regions needed for full biologic activity. We isolated a nonfunctional factor IX protein designated factor IXEagle Rock (IXER) from a patient with hemophilia B. The variant protein is indistinguishable from normal factor IX (IXN) in its migration on sodium dodecyl sulfate-gel electrophoresis, isoelectric point in urea, carbohydrate content and distribution, number of gamma-carboxyglutamic acid residues, and beta-OH aspartic acid content, and in its binding to an anti-IXN monoclonal antibody which has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. Further, IXER is cleaved to yield a factor IXa-like molecule by factor XIa/Ca2+ at a rate similar to that observed for IXN. However, in contrast to IXaN, IXaER does not bind to antithrombin-III (specific inhibitor of IXaN) and does not catalyze the activation of factor X (substrate) to factor Xa. To identify the mutation in IXER, all eight exons of IXN and IXER gene were amplified by the polymerase chain reaction technique and cloned. A single point mutation (G----T) which results in the replacement of Val for Gly363 in the catalytic domain of IXER was identified. Gly363 in factor IXa corresponds to the universally conserved Gly193 in the active site sequence of the chymotrypsin serine protease family. X-ray crystallographic data in the literature demonstrate a critical role of this Gly in stabilizing the active conformation of chymotrypsin/trypsin in two major ways: 1) in the formation of the substrate binding site; and 2) in the development of the oxyanion hole. Our computer structural data support a concept that the Gly363----Val change prevents the development of the active site conformation in factor IXa such that the substrate binding site and the oxyanion hole are not formed in the mutated enzyme.     

EFFECT OF DIMETHYL SULFOXIDE ON ZINC65 UPTAKE,RESPIRATION, AND RNA AND PROTEIN METABOLISM IN BEAN (PHASEOLUS VULGARIS) TISSUES

The effect of dimethyl sulfoxide (DMSO) on zinc⁶⁵ uptake, respiration, RNA, and protein metabolism in various tissues of two bean (Phaseolus vulgaris L.) cultivars showing differential growth responses to zinc has been studied. At a concentration of 1%, DMSO stimulated zinc uptake in excised roots, stem-callus tissue, leaf disks, and enzymically isolated leaf cells, but did not significantly alter the uptake and incorporation of C¹⁴-uracil into RNA and C¹⁴-methionine into protein, although a slight inhibition was discernible in some tissues. At a higher concentration (10%) DMSO increased Zn⁶⁵ uptake in excise roots incubated for 2 hr; however, at the same concentration, C¹⁴-uracil and C¹⁴-methionine uptake and incorporation were considerably inhibited in all the tissues. Oxygen uptake as measured with Warburg manometers was impaired, and the inhibition showed a time and concentration dependency. The fact that DMSO inhibited respiration and RNA and protein metabolism, while at the same concentration zinc uptake was increased, suggests that zinc uptake in beans is primarily a non-metabolic process. The possible mechanisms of DMSO action are discussed in the light of its reported effects on membrane permeability and cell metabolism.     

Stoichiometry of binding of high molecular weight kininogen to factor XI/XIa

Factor XI is a dimeric protein and circulates in plasma complexed with high molecular weight kininogen (HMWK). We investigated the binding of HMWK to factor XIa utilizing two active site directed fluorescent probes: nitrobenzoxadiazole aminopentyl methylphosphonofluoridate for serine and dansyl-glu-gly-arg-chloromethyl ketone for histidine. In the presence of saturating amounts of HMWK, the fluorescence of factor XIa-fluorophore was quenched by approximately 28% for each probe. Titrations of the fluorescent factor XIa with HMWK revealed that each subunit of factor XIa binds one molecule of HMWK with a Kd approximately 3.4 X 10(-8)M.     

Differential inhibition of adenosine deaminase deficient peripheral blood lymphocytes and lymphoid line cells by deoxyadenosine and adenosine.

The response to mitogens of peripheral blood lymphocytes (PBL) from adenosine deaminase deficient (ADA^?) patients with Severe Combined Immunodeficiency (SCID), but not from normals, was more sensitive to inhibition by deoxyadenosine than by adenosine. In contrast, proliferation of long-term lymphoid line (B) cells from these patients was essentially equally inhibited by adenosine and deoxyadenosine. Deoxycytidine could “rescue” ADA^? PBLs from deoxyadenosine toxicity.