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The effect of wheelchair handrim tube diameter on propulsion efficiency and force application (tube diameter and efficiency in wheelchairs).

To determine the optimum tube diameter of a standard handrim-propelled wheelchair, the effect of tube size and shape on physiological and kinetic parameters was studied. Six able-bodied male subjects performed two tests on a wheelchair ergometer. Tests were performed against work loads comparable to every day use and with two different handrim tube diameters, a handrim with an oval 25 by 30 mm diameter (LR) and one with an 18 mm diameter (SR). The large tube diameter (LR) yielded slightly but significantly lower values for the physiological parameters. Gross mechanical efficiency was on average 7% for the LR and 6.3% for the SR. No significant results were found for force application parameters related to the direction of the applied force or the torque by the hand onto the handrim surface. As technique parameters could not explain the higher mechanical efficiency (ME) when using the LR, it is suggested that hand grip constraints in the push phase (finger flexor activity) might be responsible. Another possible explanation is that with a better hand grip using LR, less stabilization by the larger muscle groups at the elbow and shoulder is needed. The measured technique parameters seem to be determined by geometric constraints of the arm and shoulder. The technique requirements resulting from the forced trajectory of the propulsion movement are also likely to determine the technique parameters. Regarding the low mechanical efficiency of handrim propulsion, which is partly caused by the forced unfavorable trajectory of the hand, an alternative propulsion mechanism is suggested.

Adult↗

Rotary dialysis: its application to the preparation of large liposomes and large proteoliposomes (protein-lipid vesicles) with high encapsulation efficiency and efficient reconstitution of membrane proteins.

An apparatus for rotary dialysis is introduced and described in detail. The component parts are inexpensive, widely available, and relatively easy to modify and assemble. The apparatus achieves increased mixing of the contents of dialysis bags by constant end-over-end rotation. This technique is particularly useful in systems where maximum contact is desired between substances which would tend to partition under standard dialysis conditions. We have applied rotary dialysis to two liposome production methods. These are (i) the calcium-EDTA-chelation method of Papahadjopoulos et al. (1), which produces large unilamellar liposomes from negatively charged phospholipids, and (ii) a procedure for the reconstitution of membrane proteins into liposomes with a large internal aqueous space, which we have developed using the calcium-EDTA-chelation technique as a point of departure. In both techniques, vesicle formation occurs when a calcium-phospholipid precipitate is dissolved by the addition of EDTA. Instead of adding a 150 mM EDTA solution directly, as described in the original method, we have used overnight rotary dialysis against buffer containing 10 mM EDTA at the vesicle formation stage. Materials are encapsulated within the aqueous interior of the vesicles at much higher efficiencies when rotary dialysis is used in either method, compared to efficiencies obtained with direct addition of EDTA (up to 37% of added material vs a maximum published efficiency of 10% for direct addition). Rotary dialysis also promotes the reconstitution of a higher proportion of the membrane proteins present in the dialysis mixture into the bilayer of large liposomes (79 vs 41.6%). It also affects the content of liposomes qualitatively, allowing better reconstitution of the Sendai virus F glycoprotein than does direct addition of EDTA. These effects may be due to the slow time course, the extensive mixing of components, and the low volume-to-phospholipid ratios maintained during vesicle formation.

Calcium↗

Thymic pathogenicity of an HIV-1 envelope is associated with increased CXCR4 binding efficiency and V5-gp41-dependent activity, but not V1/V2-associated CD4 binding efficiency and viral entry.

We previously described a thymus-tropic HIV-1 envelope (R3A Env) from a rapid progressor obtained at the time of transmission. An HIV-1 molecular recombinant with the R3A Env supported extensive replication and pathogenesis in the thymus and did not require Nef. Another Env from the same patient did not display the same thymus-tropic pathogenesis (R3B Env). Here, we show that relative to R3B Env, R3A Env enhances viral entry of T cells, increases fusion-induced cytopathicity, and shows elevated binding efficiency for both CD4 and CXCR4, but not CCR5, in vitro. We created chimeric envelopes to determine the region(s) responsible for each in vitro phenotype and for thymic pathogenesis. Surprisingly, while V1/V2 contributed to enhanced viral entry, CD4 binding efficiency, and cytopathicity in vitro, it made no contribution to thymic pathogenesis. Rather, CXCR4 binding efficiency and V5-gp41-associated activity appear to independently contribute to thymic pathogenesis of the R3A Env. These data highlight the contribution of unique HIV pathogenic factors in the thymic microenvironment and suggest that novel mechanisms may be involved in Env pathogenic activity in vivo.

CD4 Antigens↗

Mismatch repair by efficient nick-directed, and less efficient mismatch-specific, mechanisms in homologous recombination intermediates in Chinese hamster ovary cells.

Repair of single-base mismatches formed in recombination intermediates in vivo was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks (DSBs) introduced into regions of shared homology in pairs of plasmid substrates heteroallelic at 11 phenotypically silent mutations. Recombination was expected to occur primarily by single-strand annealing, yielding predicted heteroduplex DNA (hDNA) regions with three to nine mismatches. Product spectra were consistent with hDNA only occurring between DSBs. Nicks were predicted on opposite strands flanking hDNA at positions corresponding to original DSB sites. Most products had continuous marker patterns, and observed conversion gradients closely matched predicted gradients for repair initiated at nicks, consistent with an efficient nick-directed, excision-based mismatch repair system. Discontinuous patterns, seen in approximately 10% of products, and deviations from predicted gradients provided evidence for less efficient mismatch-specific repair, including G-A-->G-C specific repair that may reflect processing by a homologue of Escherichia coli MutY. Mismatch repair was > 80% efficient, which is higher than seen previously with covalently closed, artificial hDNA substrates. Products were found in which all mismatches were repaired in a single tract initiated from one or the other nick. We also observed products resulting from two tracts of intermediate length initiated from two nicks.

Animals↗

Efficient RNA interference depends on global context of the target sequence: quantitative analysis of silencing efficiency using Eulerian graph representation of siRNA.

Several aspects of gene silencing by small interfering RNA duplexes (siRNA) influence the efficiency of the silencing. They can be divided into two categories, one covering the cell-specific factors and the other covering molecular factors of the RNA interference (RNAi). A prerequisite for sequence-based siRNA design is that hybridization thermodynamics is the dominant factor. Our assumption is that cell-specific parameters (cell line, degradation, cross-hybridization, target conformation, etc.) can be pooled into an average cellular factor. Our hypothesis is that the molecular basis of the positional dependence of siRNA-induced gene silencing is the uniqueness of context of a corresponding target sequence segment relative to all other such segments along the attacked RNA. We encode this context into descriptors derived from Eulerian graph representation of siRNAs and show that the descriptor based upon the contextual similarity and predicted thermodynamic stability correlates with the experimentally observed silencing efficiency of human lamin A/C gene. We further show that information encoded in this regression function is generalizable and can be used as a predictor of siRNA efficiency in unrelated genes (CD54 and PTEN). In summary, our method represents an evolution of siRNA design from the currently used algorithms which are only qualitative in nature.

Base Sequence↗

Efficient CRISPR/Cas-SF01 genome editing tools with high editing efficiency in allotetraploid oilseed rape.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 has been widely utilized for plant genome editing, but the protospacer adjacent motif (PAM) requirement limits its editing scope. CRISPR/Cas12i3 belongs to the type-VI Cas system that has gained extensive attention due to its smaller size and less restricted canonical TTN PAM sequence. In this study, we explored the newly developed Cas-SF01 system (Cas12i3 variant) for genome editing in oilseed rape. We established an efficient protoplast transformation system in oilseed rape to compare editing efficiency between Cas-SF01 and Cas9. Cas-SF01 shows cleavage activities at the tested 5'-TTN-3' PAM sites with editing outcomes sharing considerable similarities with the CRISPR-Cas9 system in protoplast. Cas-SF01 also induces high efficiency mutagenesis for multiple target sites in stable transformed oilseed rape lines, generating mutants with multilocular silique and male sterile phenotypes. Furthermore, Cas-SF01-derived cytosine base editors (CBEs) were developed to produce targeted C-to-T base edits. Compared to SpCas9, Cas-SF01 has an expanded PAM range and effectively recognizes TTN PAMs, which has substantially broadened the scope of editable sites within the rapeseed genome. No mutations were identified at the putative off-target sites among the edited plants. This study developed a robust, first-of-its-kind Cas12 system in the allotetraploid Brassica napus, expanding the scope of editing and enriching genome-editing toolkits for biological research and genetic improvement.

Brassica napus↗

The developmentally regulated shift from membrane to secreted mu mRNA production is accompanied by an increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency.

To determine whether there are any developmental changes in the efficiencies of cleavage-polyadenylation or splicing reactions that could affect the usage of weak (suboptimal) processing signals and thus provide a basis for the regulated production of mu m versus mu s mRNA during B-lymphocyte maturation, we studied the expression of transfected mu genes in which the natural competition between cleavage-polyadenylation and splicing was replaced by alternative usage of tandem weak and strong poly(A) sites or by competition between suboptimal and optimal 5' splice junctions. Our results indicate that there is a 50 to 100% increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency as maturation proceeds from the B-cell to plasma cell stage.

Adenovirus Early Proteins↗

Vascular endothelial growth factor is efficiently synthesized in spite of low transfection efficiency of pSG5VEGF plasmids in vascular smooth muscle cells.

The limitation of lipotransfection with plasmid vectors is its low efficiency and the short-term expression of introduced genes. This is particularly important when the synthesis of high amounts of therapeutic products is required. However, growth factors with paracrine action overcome this problem. The aim of our study was to check whether the amounts of vascular endothelial growth factor (VEGF) generated after plasmid lipotransfection into vascular smooth muscle cells (VSMC) can be sufficient to stimulate endothelial cell proliferation. Two plasmids, pSG5-VEGF121 and pSG5-VEGF165, harboring human VEGF121 and VEGF165 isoforms were constructed and lipotransfected into COS-7 cells or to rat VSMC. The transfection efficiency, estimated by the expression of control, beta-galactosidase gene, was about 50% in COS-7 but rarely exceeded 5% in VSMC. However, despite this, the smooth muscle cells generated high amounts of VEGF protein, up to 3 ng/ml medium. The biological activity of this VEGF was confirmed by enhanced proliferation of human umbilical vein and coronary artery endothelial cells, stimulated with conditioned media of pSG5-VEGF transfected cells. Thus, the low transfection efficiency does not preclude the generation of high amounts of VEGF by VSMC. After reaching the maximum at about 48 h after transfection, the generation of VEGF decreased in the following days. Such a situation may be sufficient for the gene therapy of restenosis when the long-term expression of therapeutic gene(s) is not necessary. Thus, we suggest that the pSG5-VEGF121, and pSG5-VEGF165 plasmids can be used for therapeutic application.

Animals↗

[Analysis of the efficiency of transcervical resection of endometrium for treating dysfunctional uterine bleeding and factors reducing the efficiency of the operation].

OBJECTIVE: To investigate the efficiency and factors related to the recurrence of transcervical resection of endometrium (TCRE) in treating women with dysfunctional uterine bleeding (DUB). METHODS: Two hundred and twenty women with DUB were selected for TCRE. Specimens from removed endometrium were obtained during the procedures and their menstruation and menorrhagia after the operations were followed up. We also studied the uterine specimens obtained from the recurrent cases undergoing hysterectomies. The specimens were stained by hematoxylin-eosin and immuno-histochemistry respectively to evaluate the depth of removed myometrium and the histopathologic changes in relapsed cases. RESULTS: The follow up period after operation was 24 to 114 months. (1) Overall effective rate was 94.5%, in which amenorrhea rate was 25.9%, menstruation reduction rate was 68.6%. (2) The effective rate of corrective anemia was 97.3% and the satisfaction rate for the operation was 92.3%. (3) Thirty-eight cases required subsequent treatment as a result of recurrence, of which 10 cases underwent hysterectomy and 3 cases underwent repeat TCRE and 25 cases were given medicine treatment. (4) The average depth of myometrium in removed endometrium strip was 2.12 approximately 3.26 mm. (5) Endometrium regrowth was seen in the resected uterine specimens from relapsed cases and adenomyosis was also found in the intra-uterine wall in some cases. CONCLUSIONS: (1) TCRE is a safe and effective alternative treatment for DUB. (2) The main factor reducing the efficiency is either incomplete removal of endometrium or adenomyosis. (3) Standardizing the procedures and strengthening postoperative management are essential requirements for improving the efficiency of TCRE.

Adult↗

A novel nozzle for more efficient sperm orientation to improve sorting efficiency of X and Y chromosome-bearing sperm.

Efficient high-resolution detection of DNA for flow cytometric sorting of X and Y chromosome-bearing sperm is dependent on effectively orientating the sperm head to the laser beam in orthogonally configured flow systems. Normally, a beveled needle is required to enlarge the fraction of properly orientated sperm (flat side facing the laser beam). In this report, a modification to a standard jet-in-air nozzle for improved sperm orientation is presented. Inside the modified nozzle (novel nozzle), orientation forces are applied lower in the nozzle than in the current beveled injection needle system. The nozzle was tested with sperm heads from several species. This study shows that use of the nozzle to orientate cattle, swine, rabbit, mouse, and human sperm effectively improves the percentage of sperm that are properly oriented. The percentage of sperm heads oriented by use of the former system (beveled needle) ranges around 30% for most species. With the newly designed nozzle, that percentage ranges around 60%. At least a twofold increase in analysis is achieved. It was found that, unlike results with the beveled needle, the percentage of properly oriented sperm was independent of the sample rate. The introduced nozzle is a significant improvement over the beveled needle system for the analysis and sorting of sperm on the basis of DNA content. In addition to the improvement in sorted sperm production brought about by the novel nozzle when fitted to standard-speed cell sorters, it clearly also has significant potential for improving the efficiency of the Beltsville Sperm Sexing Technology for separating X and Y chromosome-bearing sperm when adapted to high-speed cell-sorting systems.

Animals↗

Efficient fine mapping of the naked caryopsis gene ( nud) by HEGS (High Efficiency Genome Scanning)/AFLP in barley.

The hulled or naked caryopsis character of barley ( Hordeum vulgare L.) is an important trait for edibility and to follow its domestication process. A single recessive gene, nud, controls the naked caryopsis character, and is located on the long arm of chromosome 7H. To develop a fine map around the nud locus efficiently, the HEGS (High Efficiency Genome Scanning) electrophoresis system was combined with amplified fragment length polymorphism (AFLP). From bulked segregant analysis of 1,894 primer combinations, 12 AFLP fragments were selected as linked markers. For mapping, an F(2 )population of 151 individuals derived from a cross between Kobinkatagi (naked type) and Triumph (hulled type) was used. Seven AFLP markers were localized near the nud region. A fine map was developed with one-order higher resolution than before, along with the seven anchor markers. Among the seven linked AFLP markers (KT1-7), KT1, KT2 and KT6 were co-dominant, and the former two were detected for their single-nucleotide polymorphisms (SNPs) in the same length of fragments after electrophoresis with the non-denaturing gels of HEGS. The nud locus has co-segregated with KT3 and KT7, and was flanked by KT2 and KT4, at the 0.3-cM proximal and the 1.2-cM distal side, respectively. Four of these AFLP markers were converted into sequence-characterized amplified region (SCAR) markers, one of which was a dominant marker co-segregating with the nud gene.

Chromosome Mapping↗

Convenient dioxin measuring method using an efficient sampling train, an efficient HPLC system and a highly sensitive HRGC/LRMS with a PTV injector.

An efficient sampling method for dioxins from flue gas, an efficient automatic purification and fractionation method by a new HPLC system using a sulfuric acid-silica/silica column, a Nitro column and a PGC column, and sensitive determination method by an economical HRGC/LRMS using a programmable temperature vaporization (PTV) injector were developed. It was confirmed that the dioxins could be easily collected by the proposed simple sampling train consisting of only two bottles, and the extracted sample could be purified sufficiently and separated as mono-ortho PCBs, non-ortho PCBs and PCDDs/PCDFs completely with the proposed HPLC system. The peak areas of GC/MS were increased linearly with the injection volume up to 40 microl by the PTV technique, and the sensitivity could be increased to 20 times higher than usual. This convenient measuring method can drastically reduce operation time, consumption of hazardous solvent and cost.

Air Pollutants↗

Polarizing a hydrophobic cavity for the efficient binding of organic guests: the case of calix[6]tren, a highly efficient and versatile receptor for neutral or cationic species.

The host-guest properties of calix[6]tren 1 have been evaluated. The receptor is based on a calix[6]arene that is covalently capped at the narrow rim by a tren unit. As a result, the system presents a concave hydrophobic cavity with, at its bottom, a grid-like nitrogenous core. Despite its well-defined cavity and opening to the outside at the large rim, 1 did not behave as a good receptor for neutral molecules in chloroform. However, it exhibited efficient endo-complexation of ammonium guests. By contrast, the per-protonated host, 1.4H(+), behaved as a remarkable receptor for small organic molecules. The complexation is driven by a strong charge-dipole interaction and hydrogen bonds between the polar guest and the tetracationic cap of the calixarene. Finally, coordination of Zn(2+) to the tren core led to the asymmetrization of calixarene cavity and to the strong but selective endo-binding of neutral ligands. This study emphasizes the efficiency of a receptor presenting a concave hydrophobic cavity that is polarized at its bottom. The resulting combination of charge-dipole, hydrogen bonding, CH-pi, and van der Waals interactions highly stabilizes the supramolecular architectures. Also, importantly, the tren cap allows the tuning of the polarization, offering either a basic (1), a highly charged and acidic (1.4H(+)), or a coordination (1.Zn(2+)) site. As a result, the system proved to be highly versatile, tunable, and interconvertible in solution by simple addition of protons, bases, or metal ions.

Journal Article↗

A transforming marker that produces merodiploids with high efficiency and stable transformants with low efficiency in Streptococcus.

A mutation (ery-r8) conferring a high level of resistance to erythromycin in the Challis strain of Streptoccus sanguis can be transferred to wild-type erythromycin-sensitive recipients via single molecules of donor DNA. The transformants thus produced are of two types: (1) cells slightly more resistant to erythromycin than wild-type and capable of segregating (at a frequency of 2 X 10(-4)/bacterium/generation) either wild-type or highly-resistant cells like the original donor type; (2) cells phenotypically and genotypically identical to the original donor type. The unstable diploids (ery-r8/+) occur with a frequency equivalent to that obtained with high-efficiency (HE) markers, whereas the stable donor-type (ery-r8) transformants occur with about five hundred times lower frequency. Penetration of the wild-type recipient by more than one molecule of DNA bearing the ery-r8 marker increases by as much as seven times the incidence of stable transformants. UV-irradiation of molecules bearing the ery-r8 marker diminishes their ability to cooperate in producing a stable transformant, although the UV sensitivity of stable transformant production by a single DNA molecule is not different from that of diploid production. Hence, stable transformants do not appear to be produced by a process typical of low efficiency (LE) markers, which are generally highly sensitive to ultraviolet irradiation. Moreover, stable ery-r8 transformants are produced with equally low frequencies in strains of S. pneumoniae that discriminate (hex+) and fail to discriminate (hex--) between HE and LE markers. We postulate that all transformations by the ery-r8 marker result in ery-r8/+ diploids, and that segregation results in the infrequent stable transformants of the original donor type. This hypothesis is supported by the observations that rifampin treatment of ery-r8/+ populations increases the frequency of segregation and similar treatment of wild-type recipients under-going transformation by the ery-r8 marker increases the frequency of stable transformants.--In producing the ery-r8/+ transformant the r8 allele is integrated close to the site of its wild-type homolog, since single molecules of DNA from this transformant can be shown to carry both alleles. Segregation of either the ery-r8 or + allele is not detectably enhanced by acridine orange or thymidine deprivation.--The ery-r8 marker occurs close to a site of mutation (ery-r2) which confers erythromycin resistance upon ribosomes. When the r2 and r8 markers are jointly transferred, ery-r2-r8/+ genomes are produced in which the r2 marker is stably integrated but the r8 marker is unstably adjoined to its wild-type homolog. Thus, the duplicated region can be quite short. When the ery-r8 marker is stably integrated, the region of the marker is refractory to subsequent transformation. Markers with properties like ery-r8 are not particularly rare, being found with a frequency of about 4% among spontaneous mutations to erythromycin resistance.

Diploidy↗

SV40 T-antigen induces breast cancer formation with a high efficiency in lactating and virgin WAP-SV-T transgenic animals but with a low efficiency in ovariectomized animals.

The whey acid protein (WAP) is a major mouse milk protein and its gene expression is induced by various lactotrophic hormones (eg, estrogen, progesterone). Transgenic animals harboring the early SV40 coding region (T/t-antigen) under the transcriptional control of the WAP promoter develop breast cancer after the first lactation period. The tumor cells synthesize the SV40 T-antigen with a high efficiency indicating that WAP-SV-T expression escapes down-regulation after the lactation period. However about 5-10% of the tumors became T-antigen negative during tumor progression and WAP-SV-T expression was only demonstrable by PCR analysis. Both T-antigen positive and negative tumor cells expressed the estrogen and progesterone receptor at a comparable rate, indicating that hormone receptor levels do not determine expression of the WAP-SV-T transgene. Furthermore, WAP and WAP-SV-T gene expression are not restricted to the pregnancy-lactation period. Virgin animals also express both genes with a low efficiency and about 70% of these animals also developed T-antigen positive breast tumors. The tumor rate however was strongly reduced in ovariectomized animals, indicating that the ovary hormones play a critical role in breast cancer formation.

Animals↗

Efficiency of the tetracycline-dependent gene expression system: complete suppression and efficient induction of the rolB phenotype in transgenic plants.

We have investigated the use of the tetracycline-dependent gene expression system to regenerate and propagate tobacco plants transformed with a gene whose product--when highly expressed--interferes with regeneration and/or further reproduction. Plants transformed with the Agrobacterium rhizogenes rolB gene under the control of the tetracycline-dependent expression system were phenotypically indistinguishable from wild type owing to efficient repression of the promoter. Induction of the rolB gene with tetracycline led to high-level expression of the rolB mRNA, which resulted in extremely stunted plants with necrotic and wrinkled leaves that did not develop a floral meristem. Upon cessation of tetracycline treatment healthy shoots developed even from severely affected meristems. Data on the dose response of the rolB phenotype as a function of tetracycline concentration demonstrate that the tetracycline-dependent gene expression system can be used to modulate the manifestation of a particular phenotype.

Gene Expression Regulation↗

Tetrabutylammonium tribromide (TBATB) as an efficient generator of HBr for an efficient chemoselective reagent for acetalization of carbonyl compounds.

Acyclic and cyclic acetals of various carbonyl compounds were obtained in excellent yields under a mild reaction condition in the presence of trialkyl orthoformate and a catalytic amount of tetrabutylammonium tribromide (TBATB) in absolute alcohol. Chemoselective acetalization of an aldehyde in the presence of ketone, unsymmetrical acetal formation, shorter reaction times, mild reaction conditions, the stability of acid-sensitive protecting groups, high efficiencies, facile isolation of the desired products, and the catalytic nature of the reagent make the present methodology a practical alternative.

Journal Article↗

Efficient adaptational demethylation of chemoreceptors requires the same enzyme-docking site as efficient methylation.

The mechanistic basis of sensory adaptation and gradient sensing in bacterial chemotaxis is reversible covalent modification of transmembrane chemoreceptors, methylation, and demethylation at specific glutamyl residues in their cytoplasmic domains. These reactions are catalyzed by a dedicated methyltransferase CheR and a dedicated methylesterase CheB. The esterase is also a deamidase that creates certain methyl-accepting glutamyls by hydrolysis of glutamine side chains. We investigated the action of CheB and its activated form, phospho-CheB, on a truncated form of the aspartate receptor of Escherichia coli that was missing the last 5 aa of the intact receptor. The deleted pentapeptide is conserved in several chemoreceptors in enteric and related bacteria. The truncated receptor was much less efficiently demethylated and deamidated than intact receptor, but essentially was unperturbed for kinase activation or transmembrane signaling. CheB bound specifically to an affinity column carrying the isolated pentapeptide, implying that in the intact receptor the pentapeptide serves as a docking site for the methylesterase/deamidase and that the truncated receptor was inefficiently modified because the enzyme could not dock. It is striking that the same pentapeptide serves as an activity-enhancing docking site for the methyltransferase CheR, the other enzyme involved in adaptational covalent modification of chemoreceptors. A shared docking site raises the tantalizing possibility that relative rates of methylation and demethylation could be influenced by competition between the two enzymes at that site.

Bacterial Proteins↗