Mild obesity does not affect perinatal outcome in gestational carrier cycles.

STUDY QUESTION: Does BMI of gestational carriers (GCs) affect perinatal outcomes after embryo transfer? SUMMARY ANSWER: Overweight and class I obesity in GCs does not affect the rate of good perinatal outcomes. WHAT IS KNOWN ALREADY: The use of GCs is increasing, but uniform guidance regarding optimal BMI for GCs is lacking. Women with obesity who conceive without fertility treatment or through autologous or donor in vitro fertilization are at higher risk of adverse maternal and fetal outcomes, but data on obesity in GCs are very limited. STUDY DESIGN, SIZE, DURATION: We performed a retrospective cohort study of 1121 GC cycles from January 2015 to December 2020 at US Fertility, the largest national partnership of fertility practices in the USA. PARTICIPANTS/MATERIALS, SETTING, AND METHODS: All GC cycles performed at a large network of fertility practices were reviewed. Same-sex partners undergoing co-IVF were excluded. The primary outcome was good perinatal outcome from the first embryo transfer, defined as a singleton live birth at ≥37 weeks of gestation with birth weight between 2500 and 4000 g. Secondary outcome measures included frequencies of live birth, clinical pregnancy, miscarriage, full-term birth, low birth weight, large for gestational age, and cesarean delivery. A generalized linear model (log-binomial) was used for each to compare outcomes across BMI groups using normal BMI (20-24.9 kg/m2) as the reference group. Risk ratios and 95% CIs were estimated for each category group relative to normal BMI. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 1121 cycles in which GCs underwent first embryo transfer, of which 263 (23.5%) were in GCs with BMI >30. Demographics and reproductive history for GCs did not differ by BMI groups. The age of intended parents, use of frozen eggs, and fresh embryo transfers were higher with increasing BMI group. There were no statistically significant associations between BMI and good perinatal outcomes, live birth, clinical pregnancy, biochemical, spontaneous abortion, or low birth weight. However, among live births, higher BMI was significantly associated with birth by cesarean (P = 0.015) and large for gestational age infants (P = 0.023). LIMITATIONS, REASONS FOR CAUTION: This was a retrospective study, and there may be unmeasured confounders. The number of patients with BMI <20 or ≥35 was small, limiting the power for these groups. We were not able to assess all maternal and fetal outcomes. WIDER IMPLICATIONS OF THE FINDINGS: In this study, we did not identify any significant impact of BMI on the chances of having a good perinatal outcome. Prior research studies have been inconsistent and this is the largest study to date. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received for this work. The authors do not have any conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

On the formation of fold-type oscillation marks in the continuous casting of steel.

Asymptotic methods are employed to revisit an earlier model for oscillation-mark formation in the continuous casting of steel. A systematic non-dimensionalization of the governing equations, which was not carried out previously, leads to a model with 12 dimensionless parameters. Analysis is provided in the same parameter regime as for the earlier model, and surprisingly simple analytical solutions are found for the oscillation-mark profiles; these are found to agree reasonably well with the numerical solution in the earlier model and very well with fold-type oscillation marks that have been obtained in more recent experimental work. The benefits of this approach, when compared with time-consuming numerical simulations, are discussed in the context of auxiliary models for macrosegregation and thermomechanical stresses and strains.

Essential Bacillus subtilis genes.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

New family of regulators in the environmental signaling pathway which activates the general stress transcription factor sigma(B) of Bacillus subtilis.

Expression of the general stress regulon of Bacillus subtilis is controlled by the alternative transcription factor sigma(B), which is activated when cells encounter growth-limiting energy or environmental stresses. The RsbT serine-threonine kinase is required to convey environmental stress signals to sigma(B), and this kinase activity is magnified in vitro by the RsbR protein, a positive regulator important for full in vivo response to salt or heat stress. Previous genetic analysis suggested that RsbR function is redundant with other unidentified regulators. A search of the translated B. subtilis genome found six paralogous proteins with significant similarity to RsbR: YetI, YezB, YkoB, YojH, YqhA, and YtvA. Their possible regulatory roles were investigated using three different approaches. First, genetic analysis found that null mutations in four of the six paralogous genes have marked effects on the sigma(B) environmental signaling pathway, either singly or in combination. The two exceptions were yetI and yezB, adjacent genes which appear to encode a split paralog. Second, biochemical analysis found that YkoB, YojH, and YqhA are specifically phosphorylated in vitro by the RsbT environmental signaling kinase, as had been previously shown for RsbR, which is phosphorylated on two threonine residues in its C-terminal region. Both residues are conserved in the three phosphorylated paralogs but are absent in the ones that were not substrates of RsbT: YetI and YezB, each of which bears only one of the conserved residues; and YtvA, which lacks both residues and instead possesses an N-terminal PAS domain. Third, analysis in the yeast two-hybrid system suggested that all six paralogs interact with each other and with the RsbR and RsbS environmental regulators. Our data indicate that (i) RsbR, YkoB, YojH, YqhA, and YtvA function in the environmental stress signaling pathway; (ii) YtvA acts as a positive regulator; and (iii) RsbR, YkoB, YojH, and YqhA collectively act as potent negative regulators whose loss increases sigma(B) activity more than 400-fold in unstressed cells.

YkdA and YvtA, HtrA-like serine proteases in Bacillus subtilis, engage in negative autoregulation and reciprocal cross-regulation of ykdA and yvtA gene expression.

HtrA-type serine proteases participate in folding and degradation of aberrant proteins and in processing and maturation of native proteins. Mutation of the corresponding genes often confers a pleiotropic phenotype that can include temperature sensitivity, sensitivity to osmotic and oxidative stress, and attenuated virulence. There are three HtrA-type serine proteases, YkdA, YvtA, and YycK, encoded in the Bacillus subtilis genome. In this report we show that YkdA and YvtA display many similarities: their expression profiles during the growth cycle in wild-type and mutant backgrounds are very alike, with expression being directed by very similar promoters. Both are induced by temperature upshift and by heterologous amylases at the transition phase of the growth cycle. These characteristics are quite different for YycK, suggesting that it has a cellular function distinct from that of the other two proteases or that it performs the same function but under different conditions. We also show that inactivation of either ykdA or yvtA results in compensating overexpression of the other gene, especially during stress conditions, with a concomitant increase in resistance to heat and hydrogen peroxide stresses. Mutation of both ykdA and yvtA leads to growth defects and to thermosensitivity. The fact that their expression increases dramatically at the transition phase of the growth cycle under certain conditions suggests that the YkdA and YvtA proteases may function in the processing, maturation, or secretion of extracellular enzymes in B. subtilis.

The missing organic molecules on Mars.

GC-MS on the Viking 1976 Mars missions did not detect organic molecules on the Martian surface, even those expected from meteorite bombardment. This result suggested that the Martian regolith might hold a potent oxidant that converts all organic molecules to carbon dioxide rapidly relative to the rate at which they arrive. This conclusion is influencing the design of Mars missions. We reexamine this conclusion in light of what is known about the oxidation of organic compounds generally and the nature of organics likely to come to Mars via meteorite. We conclude that nonvolatile salts of benzenecarboxylic acids, and perhaps oxalic and acetic acid, should be metastable intermediates of meteoritic organics under oxidizing conditions. Salts of these organic acids would have been largely invisible to GC-MS. Experiments show that one of these, benzenehexacarboxylic acid (mellitic acid), is generated by oxidation of organic matter known to come to Mars, is rather stable to further oxidation, and would not have been easily detected by the Viking experiments. Approximately 2 kg of meteorite-derived mellitic acid may have been generated per m(2) of Martian surface over 3 billion years. How much remains depends on decomposition rates under Martian conditions. As available data do not require that the surface of Mars be very strongly oxidizing, some organic molecules might be found near the surface of Mars, perhaps in amounts sufficient to be a resource. Missions should seek these and recognize that these complicate the search for organics from entirely hypothetical Martian life.

Expression of ykdA, encoding a Bacillus subtilis homologue of HtrA, is heat shock inducible and negatively autoregulated.

There are three members of the HtrA family of serine proteases, YkdA, YvtA, and YyxA, encoded in the chromosome of Bacillus subtilis. In this study, we report on the promoter structure and regulation of ykdA expression. The ykdA gene is heat inducible, exhibiting a biphasic pattern of expression during a 60-min interval after heat shock. Increased expression after heat shock occurs at the transcriptional level. The heat-shock-inducible promoter has a single mismatch with a SigA-type -10 motif, but does not exhibit similarity to a SigA -35 region. There are six octamer repeats with a consensus TTTTCACA positioned at, and upstream of, the normal position of a -35 region. While repeats V and VI appear dispensable, repeat IV is essential for normal thermoinducible expression. This promoter structure is also found in the control region of yvtA, encoding a second member of this family of proteases. Expression of ykdA is negatively autoregulated both during the growth cycle and during heat shock. Our evidence suggests that YkdA protease activity is not required for this form of regulation. Null mutants of ykdA display increased tolerance to heat and are 80-fold more resistant to 10 mM hydrogen peroxide than wild-type cells. However, ykdA expression is not induced by hydrogen peroxide. These results indicate that the regulon to which YkdA belongs is linked to the oxidative stress response in B. subtilis.

ClpE, a novel type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis.

Clp ATPases, which include the ubiquitous HSP100 family, are classified according to their structural features and sequence similarities. During the course of the Bacillus subtilis genome sequencing project, we identified a gene encoding a new member of the HSP100 family. We designated this protein ClpE, as it is the prototype of a novel subfamily among the Clp ATPases, and have identified homologues in several bacteria, including Listeria monocytogenes, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, Lactobacillus sakei and Clostridium acetobutylicum. A unique feature of these Hsp100-type Clp ATPases is their amino-terminal zinc finger motif. Unlike the other class III genes of B. subtilis (clpC and clpP ), clpE does not appear to be required for stress tolerance. Transcriptional analysis revealed two sigmaA-type promoters, expression from which was shown to be inducible by heat shock and puromycin treatment. Investigation of the regulatory mechanism controlling clpE expression indicates that this gene is controlled by CtsR and is thus a member of the class III heat shock genes of B. subtilis. CtsR negatively regulates clpE expression by binding to the promoter region, in which five CtsR binding sites were identified through DNase I footprinting and sequence analysis.

Proteome composition and codon usage in spirochaetes: species-specific and DNA strand-specific mutational biases.

The genomes of the spirochaetes Borrelia burgdorferi and Treponema pallidum show strong strand-specific skews in nucleotide composition, with the leading strand in replication being richer in G and T than the lagging strand in both species. This mutation bias results in codon usage and amino acid composition patterns that are significantly different between genes encoded on the two strands, in both species. There are also substantial differences between the species, with T.pallidum having a much higher G+C content than B. burgdorferi. These changes in amino acid and codon compositions represent neutral sequence change that has been caused by strong strand- and species-specific mutation pressures. Genes that have been relocated between the leading and lagging strands since B. burgdorferi and T.pallidum diverged from a common ancestor now show codon and amino acid compositions typical of their current locations. There is no evidence that translational selection operates on codon usage in highly expressed genes in these species, and the primary influence on codon usage is whether a gene is transcribed in the same direction as replication, or opposite to it. The dnaA gene in both species has codon usage patterns distinctive of a lagging strand gene, indicating that the origin of replication lies downstream of this gene, possibly within dnaN. Our findings strongly suggest that gene-finding algorithms that ignore variability within the genome may be flawed.

Base composition skews, replication orientation, and gene orientation in 12 prokaryote genomes.

Variation in GC content, GC skew and AT skew along genomic regions was examined at third codon positions in completely sequenced prokaryotes. Eight out of nine eubacteria studied show GC and AT skews that change sign at the origin of replication. The leading strand in DNA replication is G-T rich at codon position 3 in six eubacteria, but C-T rich in two Mycoplasma species. In M. genitalium the AT and GC skews are symmetrical around the origin and terminus of replication, whereas its GC content variation has been shown to have a centre of symmetry elsewhere in the genome. Borrelia burgdorferi and Treponema pallidum show extraordinary extents of base composition skew correlated with direction of DNA replication. Base composition skews measured at third codon positions probably reflect mutational biases, whereas those measured over all bases in a sequence (or at codon positions 1 and 2) can be strongly affected by protein considerations due to the tendency in some bacteria for genes to be transcribed in the same direction that they are replicated. Consequently in some species the direction of skew for total genomic DNA is opposite to that for codon position 3.

One of two osmC homologs in Bacillus subtilis is part of the sigmaB-dependent general stress regulon.

In this report we present the identification and analysis of two Bacillus subtilis genes, yklA and ykzA, which are homologous to the partially RpoS-controlled osmC gene from Escherichia coli. The yklA gene is expressed at higher levels in minimal medium than in rich medium and is driven by a putative vegetative promoter. Expression of ykzA is not medium dependent but increases dramatically when cells are exposed to stress and starvation. This stress-induced increase in ykzA expression is absolutely dependent on the alternative sigma factor sigmaB, which controls a large stationary-phase and stress regulon. ykzA is therefore another example of a gene common to the RpoS and sigmaB stress regulons of E. coli and B. subtilis, respectively. The composite complex expression pattern of the two B. subtilis genes is very similar to the expression profile of osmC in E. coli.

Lysis genes of the Bacillus subtilis defective prophage PBSX.

Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they are xepA, encoding an exported protein; xhlA, encoding a putative membrane-associated protein; xhlB, encoding a putative holin; and xlyA, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of Bacillus subtilis 168. The results show that xepA is unlikely to be involved in host cell lysis. Expression of both xhlA and xhlB is necessary to effect host cell lysis of B. subtilis. Expression of xhlB (encoding the putative holin) together with xlyA (encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when xlyA is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Expression of AbrB, a transition state regulator from Bacillus subtilis, is growth phase dependent in a manner resembling that of Fis, the nucleoid binding protein from Escherichia coli.

The transition state regulator AbrB functions as an activator, a repressor, and a preventer of gene expression in Bacillus subtilis. In this paper, we show that expression of abrB is growth phase dependent. Accumulation of abrB transcript is restricted to a short period spanning the transition between the lag and exponential phases of the growth cycle. The level of abrB transcript then falls sharply, and transcript cannot be detected at the mid-exponential period of the growth cycle. The level of AbrB protein is also maximal during early exponential growth but decreases gradually throughout the remainder of the growth cycle. The abrupt reduction of abrB transcript level during the early period of the growth cycle is effected by the phosphorylated form of the response regulator Spo0p3and to a lesser extent by negative autoregulation. The growth cycle-dependent expression of abrB is very similar to that observed for fis in Escherichia coli and in Salmonella typhimurium. Although AbrB and Fis are not homologous proteins, they display extensive similarity in terms of size, DNA binding characteristics, growth cycle-dependent patterns of expression, and their control over the expression of a varied group of operons. We hypothesize therefore that AbrB, like Fis, is a nucleoid binding protein.

The phage-like element PBSX and part of the skin element, which are resident at different locations on the Bacillus subtilis chromosome, are highly homologous.

PBSX and skin are two unusual genetic elements resident on the Bacillus subtilis chromosome. PBSX is a phage-like element located at approximately 100 degrees which is induced by the SOS response and results in cell lysis with the release of phage-like particles. The phage particles contain bacterial chromosomal DNA and kill sensitive bacteria without injecting DNA. The skin element is located at approximately 230 degrees on the chromosome and is positioned within the sigK open reading frame (ORF). It is excised at a particular stage of sporulation, leading to reconstitution of the complete sigK gene. In this paper, we show that there are phage-like operons present in the skin element which are highly homologous to the region of PBSX comprising part of the control region and the late operon. These operons are similar in terms of their gene organization, the percentage identity of the products of homologous ORFs and the positioning and strengths of ribosome-binding sites for each ORF. Although this high degree of conservation suggests that the phange-like operons in skin can be expressed, expression of the late operon was not detected during exponential growth, during sporulation or after induction of the SOS response. However two non-phage-like operons in the skin element are expressed and have distinct expression profiles that are dependent on the growth and developmental status of the cell.