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Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.

eIF2B is a five-subunit guanine nucleotide exchange factor that is negatively regulated by phosphorylation of the ␣ subunit of its substrate, eIF2, leading to inhibition of translation initiation. To analyze this regulatory mechanism, we have characterized 29 novel mutations in the homologous eIF2B subunits encoded by GCD2, GCD7, and GCN3 that reduce or abolish inhibition of eIF2B activity by eIF2 phosphorylated on its ␣ subunit [eIF2(␣P)]. Most, if not all, of the mutations decrease sensitivity to eIF2(␣P) without excluding GCN3, the nonessential subunit, from eIF2B; thus, all three proteins are critical for regulation of eIF2B by eIF2(␣P). The mutations are clustered at both ends of the homologous region of each subunit, within two segments each of approximately 70 amino acids in length. Several mutations alter residues at equivalent positions in two or all three subunits. These results imply that structurally similar segments in GCD2, GCD7, and GCN3 perform related functions in eIF2B regulation. We propose that these segments form a single domain in eIF2B that makes multiple contacts with the ␣ subunit of eIF2, around the phosphorylation site, allowing eIF2B to detect and respond to phosphoserine at residue 51. Most of the eIF2 is phosphorylated in certain mutants, suggesting that these substitutions allow eIF2B to accept phosphorylated eIF2 as a substrate for nucleotide exchange.The current model for translation initiation proposes that translation initiation factor 2 (eIF2) forms a ternary complex with GTP and charged initiator tRNA Met , which binds to 40S ribosomes. After mRNA binding to the ribosome and recognition of the AUG codon by initiator tRNA Met , GTP hydrolysis releases an eIF2 ⅐ GDP binary complex. For eIF2 to participate in further rounds of initiation, it must be recycled to eIF2 ⅐ GTP by the guanine nucleotide exchange factor eIF2B. This recycling of eIF2 ⅐ GDP to eIF2 ⅐ GTP by eIF2B is inhibited by phosphorylation of eIF2 on its ␣ subunit (eIF2␣) at serine 51 (reviewed in references 30 and 44). Three protein kinases, known as HRI, PKR, and GCN2, specifically phosphorylate Ser-51 of eIF2␣ under different stress conditions (12,48). HRI is activated in mammalian reticulocytes in response to heme deprivation, whereas PKR (also known as DAI or p68 kinase) is part of the antiviral response and is activated by double-stranded RNA. Both kinases phosphorylate eIF2 to shut off total protein synthesis by complete inhibition of eIF2B.In the yeast Saccharomyces cerevisiae, GCN2 phosphorylates eIF2 in response to starvation for amino acids or purines. The level of eIF2 phosphorylated on its ␣ subunit [eIF2(␣P)] produced by moderate starvation in yeast cells is not sufficient to inhibit total protein synthesis; however, it does result in a specific increase in translation of GCN4 mRNA, encoding a transcriptional activator of amino acid biosynthetic genes (reviewed in reference 23). Thus, translation of GCN4 is inversely coupled to the concentration of eIF2 ⅐ GTP ⅐ tRNA i Met ternary complexes and, therefo...

Copper deprivation of Saccharomyces cerevisiae induces transcription of the FRE1 and CTR1 genes. FRE1 encodes a surface reductase capable of reducing and mobilizing copper chelates outside the cell, and CTR1 encodes a protein mediating copper uptake at the plasma membrane. In this paper, the protein encoded by MAC1 is identified as the factor mediating this homeostatic control. A novel dominant allele of MAC1, MAC1 up2 , is mutated in a Cys-rich domain that may function in copper sensing (a G to A change of nucleotide 812 resulting in a Cys-271 to Tyr substitution). This mutant is functionally similar to the MAC1 up1 allele in which His-279 in the same domain has been replaced by Gln. Both mutations confer constitutive copper-independent expression of FRE1 and CTR1. A sequence including the palindrome TTTGCTCA . . . TGAGCAAA, appearing within the 5-flanking region of the CTR1 promoter, is necessary and sufficient for the copper-and MAC1-dependent CTR1 transcriptional regulation. An identical sequence appears as a direct repeat in the FRE1 promoter. The data indicate that the signal resulting from copper deprivation is transduced via the Cys-rich motif of MAC1 encompassing residues 264 -279. MAC1 then binds directly and specifically to the CTR1 and FRE1 promoter elements, inducing transcription of those target genes. This model defines the homeostatic mechanism by which yeast regulates the cell acquisition of copper in response to copper scarcity or excess.

To understand how phosphorylation of eukaryotic translation initiation factor (eIF)-2␣ in Saccharomyces cerevisiae stimulates GCN4 mRNA translation while at the same time inhibiting general translation initiation, we examined the effects of altering the gene dosage of initiator tRNA Met , eIF-2, and the guanine nucleotide exchange factor for eIF-2, eIF-2B. Overexpression of all three subunits of eIF-2 or all five subunits of eIF-2B suppressed the effects of eIF-2␣ hyperphosphorylation on both GCN4-specific and general translation initiation. Consistent with eIF-2 functioning in translation as part of a ternary complex composed of eIF-2, GTP, and Met-tRNA i Met , reduced gene dosage of initiator tRNA Met mimicked phosphorylation of eIF-2␣ and stimulated GCN4 translation. In addition, overexpression of a combination of eIF-2 and tRNA i Met suppressed the growthinhibitory effects of eIF-2 hyperphosphorylation more effectively than an increase in the level of either component of the ternary complex alone. These results provide in vivo evidence that phosphorylation of eIF-2␣ reduces the activities of both eIF-2 and eIF-2B and that the eIF-2 ⅐ GTP ⅐ Met-tRNA i Met ternary complex is the principal component limiting translation in cells when eIF-2␣ is phosphorylated on serine 51. Analysis of eIF-2␣ phosphorylation in the eIF-2-overexpressing strain also provides in vivo evidence that phosphorylated eIF-2 acts as a competitive inhibitor of eIF-2B rather than forming an excessively stable inactive complex. Finally, our results demonstrate that the concentration of eIF-2 ⅐ GTP ⅐ Met-tRNA i Met ternary complexes is the cardinal parameter determining the site of reinitiation on GCN4 mRNA and support the idea that reinitiation at GCN4 is inversely related to the concentration of ternary complexes in the cell.The current model for the mechanism of translation initiation in eukaryotic cells derives from biochemical analysis of mammalian cell-free systems and characterization of individual reactions with purified initiation factors. These studies have identified eukaryotic initiation factor (eIF)-2 as the protein responsible for binding the initiator Met-tRNA (MettRNA i Met ) to the 40S ribosomal subunit in an early step of the initiation pathway (see reviews in references 26 and 31). It is believed that the Met-tRNA i Met is delivered as part of a ternary complex composed of eIF-2, GTP, and Met-tRNA i Met . After binding of the ternary complex to the 40S ribosomal subunit, the GTP is hydrolyzed to GDP and eIF-2 is released in a binary complex with GDP. Because mammalian eIF-2 has a 100-to 400-fold-higher affinity for GDP than for GTP, a guanine nucleotide exchange factor known as eIF-2B is required to recycle eIF-2 ⅐ GDP back to eIF-2 ⅐ GTP, allowing eIF-2 to function in a subsequent round of translation initiation (26,36). Phosphorylation of eIF-2␣ on serine 51 inhibits the exchange of GTP for GDP on eIF-2. Not only is exchange blocked on the phosphorylated eIF-2 molecule, but phosphorylated eIF-2 also prevents eIF-2B from re...

Background The coronavirus disease 2019 (COVID‐19) has been spreading all over the world since December 2019. However, medical information regarding the urogenital involvement in recovered COVID‐19 patients is limited or unknown. Objectives To comprehensively evaluate urogenital involvement in recovered COVID‐19 patients. Materials and methods Men aged between 20 years and 50 years who were diagnosed with SARS‐CoV‐2 infection and recovered when the study was conducted were enrolled in our study. Demographic and clinical characteristics, and history of hospitalization were collected and analyzed. Urine, expressed prostatic secretions (EPSs), and semen samples were collected for SARS‐CoV‐2 RNA detection. Semen quality and hormonal profiles were analyzed. Results Among 74 male recovered COVID‐19 patients, 11 (14.9%) were asymptomatic, classified into mild type, and 31 (41.9%) were classified into moderate type. The remaining patients (32/74, 43.2%) had severe pneumonia. No critically ill recovered COVID‐19 patient was recruited in our cohort. The median interval between last positive pharyngeal swab RT‐PCR test and semen samples collection was 80 days (IQR, 64‐93). The median age was 31 years (IQR, 27‐36; range, 21‐49), and the median body mass index (BMI) was 24.40 (IQR, 22.55‐27.30). Forty‐five (61.6%) men were married, and 28 (38.4%) were unmarried. Fifty‐three (72.6%) patients denied cigarette smoking, 18 (24.7%) were active smokers, and 2 of them were past smokers. The majority of our participants (53/74, 72.6%) did not consume alcohol. Fever occurred in most of the patients (75.3%), and 63 of them had abnormal chest CT images. Only one patient complained of scrotal discomfort during the course of COVID‐19, which was ruled out orchitis by MRI (data not shown). A total of 205 samples were collected for SARS‐CoV‐2 detection (74 urine samples, 70 semen samples, and 61 EPS samples). However, viral nucleic acid was not detected in body fluids from the urogenital system. In terms of hormonal profiles, the levels of FSH, LH, testosterone, and estradiol were 5.20 [4.23] mIU/mL, 3.95 [1.63] mIU/mL, 3.65 [1.19] ng/mL, and 39.48 [12.51] pg/mL, respectively. And these values were within the normal limits. The overall semen quality of recovered COVID‐19 patients was above the lower reference limit released by the WHO. While compared with healthy control, sperm concentration, total sperm count, and total motility were significantly declined. In addition, different clinical types of COVID‐19 have no significant difference in semen parameters, but total sperm count showed a descending trend. Interestingly, subjects with a longer recovery time showed worse data for sperm quality. Small sample size and lacking semen parameters before the infection are the major limitations of our study. Discussion and conclusions To the best of our knowledge, it is the largest cohort study with longest follow‐up for urogenital evaluation comprehensively so far. Direct urogenital involvement was not found in the recovered COVID‐19 male p...

Eukaryotic translation initiation factor 2B (eIF2B) is a five-subunit complex that catalyzes guanine nucleotide exchange on eIF2. Phosphorylation of the alpha subunit of eIF2 [creating eIF2(alphaP]) converts eIF2 x GDP from a substrate to an inhibitor of eIF2B. We showed previously that the inhibitory effect of eIF2(alphaP) can be decreased by deletion of the eIF2B alpha subunit (encoded by GCN3) and by point mutations in the beta and delta subunits of eIF2B (encoded by GCD7 and GCD2, respectively). These findings, plus sequence similarities among GCD2, GCD7, and GCN3, led us to propose that these proteins comprise a regulatory domain that interacts with eIF2(alphaP) and mediates the inhibition of eIF2B activity. Supporting this hypothesis, we report here that overexpression of GCD2, GCD7, and GCN3 specifically reduced the inhibitory effect of eIF2(alphaP) on translation initiation in vivo. The excess GCD2, GCD7, and GCN3 were coimmunoprecipitated from cell extracts, providing physical evidence that these three proteins can form a stable subcomplex. Formation of this subcomplex did not compensate for a loss of eIF2B function by mutation and in fact lowered eIF2B activity in strains lacking eIF2(alphaP). These findings indicate that the trimeric subcomplex does not possess guanine nucleotide exchange activity; we propose, instead, that it interacts with eIF2(alphaP) and prevents the latter from inhibiting native eIF2B. Overexpressing only GCD2 and GCD7 also reduced eIF2(alphaP) toxicity, presumably by titrating GCN3 from eIF2B and producing the four-subunit form of eIF2B that is less sensitive to eIF2(alphaP). This interpretation is supported by the fact that overexpressing GCD2 and GCD7 did not reduce eIF2(alphaP) toxicity in a strain lacking GCN3; however, it did suppress the impairment of eIF2B caused by the gcn3c-R104K mutation. An N-terminally truncated GCD2 protein interacted with other eIF2B subunits only when GCD7 and GCN3 were overexpressed, in accordance with the idea that the portion of GCD2 homologous to GCD7 and GCN3 is sufficient for complex formation by these three proteins. Together, our results provide strong evidence that GCN3, GCD7, and the C-terminal half of GCD2 comprise the regulatory domain in eIF2B.

This study identifies several novel susceptibility genes for MMD. The association with homocysteine metabolism and the immune system enrichment of susceptibility gene expression suggest that therapeutic interventions targeting these pathways may be effective approaches for MMD treatment.