Correction to: Comparative Analysis of the Expression of Glutathione Peroxidase and Glutathione Reductase Genes in Human Sperm after Cryopreservation.

The second author's name should read A. Yu. Romanov.

Comparative Analysis of the Expression of Glutathione Peroxidase and Glutathione Reductase Genes in Human Sperm after Cryopreservation.

We analyzed the effect of cryopreservation on the expression of glutathione peroxidase (GPX1) and glutathione reductase (GSR) genes in human sperm cells (15 sperm samples from fertile donors and 10 samples from infertile patients). The relative expression of GPX1 and GSR genes was determined by real-time PCR. The rate of post-thaw recovery was 2.1 times higher in the group of fertile donors. A significant increase in the expression of GPX1, but not GSR, was observed in sperm samples from infertile patients, while in patients with infertility, GPX1 expression significantly decreased after cryopreservation/thawing, in samples from fertile donors after the same procedure it increased to the level observed in the sperm samples from infertile patients. A positive correlation was revealed between GPX1 expression and sperm cryotolerance.

Confirming genes influencing risk to cleft lip with/without cleft palate in a case-parent trio study.

A collection of 1,108 case-parent trios ascertained through an isolated, nonsyndromic cleft lip with or without cleft palate (CL/P) was used to replicate the findings from a genome-wide association study (GWAS) conducted by Beaty et al. (Nat Genet 42:525-529, 2010), where four different genes/regions were identified as influencing risk to CL/P. Tagging SNPs for 33 different genes were genotyped (1,269 SNPs). All four of the genes originally identified as showing genome-wide significance (IRF6, ABCA4 and MAF, plus the 8q24 region) were confirmed in this independent sample of trios (who were primarily of European and Southeast Asian ancestry). In addition, eight genes classified as 'second tier' hits in the original study (PAX7, THADA, COL8A1/FILIP1L, DCAF4L2, GADD45G, NTN1, RBFOX3 and FOXE1) showed evidence of linkage and association in this replication sample. Meta-analysis between the original GWAS trios and these replication trios showed PAX7, COL8A1/FILIP1L and NTN1 achieved genome-wide significance. Tests for gene-environment interaction between these 33 genes and maternal smoking found evidence for interaction with two additional genes: GRID2 and ELAVL2 among European mothers (who had a higher rate of smoking than Asian mothers). Formal tests for gene-gene interaction (epistasis) failed to show evidence of statistical interaction in any simple fashion. This study confirms that many different genes influence risk to CL/P.

A catalog of associations between rare coding variants and COVID-19 outcomes.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes coronavirus disease-19 (COVID-19), a respiratory illness that can result in hospitalization or death. We investigated associations between rare genetic variants and seven COVID-19 outcomes in 543,213 individuals, including 8,248 with COVID-19. After accounting for multiple testing, we did not identify any clear associations with rare variants either exome-wide or when specifically focusing on (i) 14 interferon pathway genes in which rare deleterious variants have been reported in severe COVID-19 patients; (ii) 167 genes located in COVID-19 GWAS risk loci; or (iii) 32 additional genes of immunologic relevance and/or therapeutic potential. Our analyses indicate there are no significant associations with rare protein-coding variants with detectable effect sizes at our current sample sizes. Analyses will be updated as additional data become available, with results publicly browsable at https://rgc-covid19.regeneron.com.

A circulating myocardial depressant substance in humans with septic shock. Septic shock patients with a reduced ejection fraction have a circulating factor that depresses in vitro myocardial cell performance.

We have previously described a subpopulation of patients with septic shock who had a reversible depression of radionuclide-determined left ventricular ejection fraction (EF). To investigate the mechanism of this myocardial depression, an in vitro model of mammalian myocardial cell performance was established employing primary spontaneously beating rat myocardial cells. The contraction of a single cardiac cell was quantitated by recording the changes in area occupied by the cell during contraction and relaxation. In 20 septic shock patients during the acute phase, the mean left ventricular EF was decreased (mean = 0.33, normal mean = 0.50), and serum obtained during this acute phase induced a mean (+/- standard error of the mean) 33 +/- 4% decrease in extent and 25 +/- 4% decrease in velocity of myocardial cell shortening during contraction (P less than 0.001). In contrast, serum obtained from 11 of these same patients before shock (n = 2) or after recovery (n = 9) of the left ventricular EF (mean = 0.50) showed a return toward normal in extent and velocity of shortening (P less than 0.001). Sera from 17 critically ill nonseptic patients, from 10 patients with structural heart disease as a cause for a depressed EF, and from 12 healthy laboratory personnel, induced no significant changes in in vitro myocardial cell performance. In 20 patients during the acute phase of septic shock, the decreased EF in vivo demonstrated a significant correlation (r = +0.52, P less than 0.01) with a decrease in the extent of myocardial cell shortening in vitro. The quantitative and temporal correlation between the decreased left ventricular EF and this serum myocardial depressant substance argues for a pathophysiologic role for this depressant substance in producing the reversible cardiomyopathy seen during septic shock in humans.

Role of exonucleolytic degradation in group I intron homing in phage T4.

Homing of the phage T4 td intron is initiated by the intron-encoded endonuclease I-TevI, which cleaves the intronless allele 23 and 25 nucleotides upstream of the intron insertion site (IS). The distance between the I-TevI cleavage site (CS) and IS implicates endo- and/or exonuclease activities to resect the DNA segment between the IS and CS. Furthermore, 3' tails must presumably be generated for strand invasion by 5'-3' exonuclease activity. Three experimental approaches were used to probe for phage nucleases involved in homing: a comparative analysis of in vivo homing levels of nuclease-deficient phage, an in vitro assay of nuclease activity and specificity, and a coconversion analysis of flanking exon markers. It was thereby demonstrated that T4 RNase H, a 5'-3' exonuclease, T4 DNA exonuclease A (DexA) and the exonuclease activity of T4 DNA polymerase (43Exo), 3'-5' exonucleases, play a role in intron homing. The absence of these functions impacts not only homing efficiency but also the extent of degradation and flanking marker coconversion. These results underscore the critical importance of the 3' tail in intron homing, and they provide the first direct evidence of a role for 3' single-stranded DNA ends as intermediates in T4 recombination. Also, the involvement of RNase H, DexA, and 43Exo in homing provides a clear example of the harnessing of functions variously involved in phage nucleic acid metabolism for intron propagation.

Intron homing with limited exon homology. Illegitimate double-strand-break repair in intron acquisition by phage t4.

The td intron of bacteriophage T4 encodes a DNA endonuclease that initiates intron homing to cognate intronless alleles by a double-strand-break (DSB) repair process. A genetic assay was developed to analyze the relationship between exon homology and homing efficiency. Because models predict exonucleolytic processing of the cleaved recipient leading to homologous strand invasion of the donor allele, the assay was performed in wild-type and exonuclease-deficient (rnh or dexA) phage. Efficient homing was supported by exon lengths of 50 bp or greater, whereas more limited exon lengths led to a precipitous decline in homing levels. However, extensive homology in one exon still supported elevated homing levels when the other exon was completely absent. Analysis of these "one-sided" events revealed recombination junctions at ectopic sites of microhomology and implicated nucleolytic degradation in illegitimate DSB repair in T4. Interestingly, homing efficiency with extremely limiting exon homology was greatly elevated in phage deficient in the 3'-5' exonuclease, DexA, suggesting that the length of 3' tails is a major determinant of the efficiency of DSB repair. Together, these results suggest that illegitimate DSB repair may provide a means by which introns can invade ectopic sites.

Homology requirements for double-strand break-mediated recombination in a phage lambda-td intron model system.

Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage lambda model system was developed to analyze exon homology effects on intron homing and determine the role of the lambda 5'-3' exonuclease complex (Red alpha beta) in the repair event. Efficient intron homing depended on exon lengths in the 35- to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type lambda, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology.

Depressed left ventricular performance. Response to volume infusion in patients with sepsis and septic shock.

Volume infusion, to increase preload and to enhance ventricular performance, is accepted as initial management of septic shock. Recent evidence has demonstrated depressed myocardial function in human septic shock. We analyzed left ventricular performance during volume infusion using serial data from simultaneously obtained pulmonary artery catheter hemodynamic measurements and radionuclide cineangiography. Critically ill control subjects (n = 14), patients with sepsis but without shock (n = 21), and patients with septic shock (n = 21) had prevolume infusion hemodynamic measurements determined and received statistically similar volumes of fluid resulting in similar increases in pulmonary capillary wedge pressure. There was a strong trend (p = 0.004) toward less of a change in left ventricular stroke work index (LVSWI) after volume infusion in patients with sepsis and septic shock compared with control subjects. The LVSWI response after volume infusion was significantly less in patients with septic shock when compared with critically ill control subjects (p less than 0.05). These data demonstrate significantly altered ventricular performance, as measured by LVSWI, in response to volume infusion in patients with septic shock.

Right ventricular dysfunction and dilatation, similar to left ventricular changes, characterize the cardiac depression of septic shock in humans.

Septic shock in humans is usually characterized by a high cardiac output, a low systemic vascular resistance, reversible depression of left ventricular ejection fraction, and transient left ventricular dilatation. The relationship of left ventricular to right ventricular function in septic shock is poorly understood. To evaluate right ventricular vs left ventricular performance and to evaluate the relation of biventricular performance to survival, we performed serial hemodynamic and radionuclide angiographic studies in 39 patients with septic shock. Right ventricular ejection fraction was calculated using the two regions of interest method. There were 22 survivors and 17 nonsurvivors. Comparing initial with final (after recovery for survivors; within 24 hours of death for nonsurvivors) studies, each survivor's cardiovascular performance returned toward normal, with significant increases in mean arterial pressure, left and right ventricular ejection fraction, and right ventricular stroke work index. Their profiles also demonstrated significant decreases in central venous pressure, pulmonary artery wedge pressure, pulmonary artery mean pressure, and left and right ventricular end-diastolic volume indices. From initial to final study in the nonsurvivors, there was a statistically significant increase in heart rate but no change in any other cardiovascular parameter, indicating a persistence of the initial cardiovascular dysfunction until death. Comparing serial studies, the pattern of change in right vs left ventricular function was very similar (same direction in 82 percent of patients). Thus, myocardial depression in human septic shock affects both ventricles simultaneously with a similar pattern of dysfunction.

Interleukin-2 administration causes reversible hemodynamic changes and left ventricular dysfunction similar to those seen in septic shock.

Interleukin-2, a lymphocyte product, has well demonstrated antitumor activity in humans. Early clinical studies showed hemodynamic alterations in patients receiving the drug as antitumor immunotherapy. We serially assessed interleukin-2-associated hemodynamic parameters and left ventricular ejection fractions in five patients with neoplastic diseases unresponsive to conventional therapies. By day 4 of therapy, compared with baseline (preinterleukin-2), all patients developed tachycardia (p less than 0.01), decreased mean arterial blood pressure (p less than 0.05), increased cardiac index (p less than 0.05), and decreased systemic vascular resistance (p less than 0.01). In addition, left ventricular ejection fraction fell from 58.0 +/- 4.7 to 36.4 +/- 4.0 percent (0.05 less than p less than 0.10), which was associated with a trend toward left ventricular dilatation manifested by an increase in left ventricular end-diastolic volume index. Transient renal dysfunction was noted in all five patients, and one developed transient respiratory failure; both types of organ dysfunction recovered to baseline values after cessation of immunotherapy. Thus, interleukin-2 induces multiple reversible cardiovascular abnormalities that are similar to the hemodynamic manifestations of human septic shock.

A circulating myocardial depressant substance is associated with cardiac dysfunction and peripheral hypoperfusion (lactic acidemia) in patients with septic shock.

Using spontaneously beating rat myocardial cells as an in vitro model of myocardial depression, recent studies demonstrated that septic shock patients' sera frequently contain a myocardial depressant substance (MDS) that is associated with a reversible decrease in left ventricular ejection fraction (LVEF). To further characterize MDS, 50 consecutive patients with possible septic shock were studied serially from shock onset until recovery or death. Thirty-four patients had criteria diagnostic of septic shock, and 16 had a nonseptic critical illness. Of the 34, 14 met strict criteria for circulating MDS, with a mean inhibition of 35 percent (range 20 percent to 62 percent). Compared with those patients not exhibiting significant MDS activity, the 14 MDS-positive patients had a lower mean minimal EF (28 percent vs 39 percent, p less than 0.01), a greater mean decrease in EF (22.1 percent vs 8.8 percent, p = 0.002), a higher pulmonary artery wedge pressure (16.8 vs 11.9 mm Hg, p less than 0.01), greater LV dilatation (162 vs 118 ml/m2, p = 0.02), and a higher circulating mean peak lactic acid (6.9 vs 2.7 mmol/L, p less than 0.01). In the 14 MDS-positive patients, the in vitro myocardial cell depression had a negative correlation with the in vivo EF (r = -060, p less than 0.05). These findings suggest that a circulating MDS is a cause of the myocardial depression frequently accompanying human septic shock.

Ultrasound guidance improves the success rate of internal jugular vein cannulation. A prospective, randomized trial.

STUDY OBJECTIVE: To compare conventional versus ultrasound-guided internal jugular vein cannulation techniques. DESIGN: Patients were randomly assigned to receive either conventional or two-dimensional ultrasound-guided internal jugular vein cannulation. Patients who could not be cannulated with five or fewer passes by either technique, were crossed over to the other technique. SETTING: Clinical research unit in a tertiary care center. PATIENTS: All consecutive patients who required urgent or urgent-elective internal jugular vein cannulation during the study period. INTERVENTIONS: The two-dimensional ultrasound transducer imaged all cannulation attempts. For patients randomized to ultrasound guidance, the operator viewed two-dimensional ultrasound images, and received verbal guidance from the ultrasound technician. For patients randomized to the conventional arm, two-dimensional ultrasound images were recorded without visual or verbal feedback. MEASUREMENTS AND MAIN RESULTS: Two-dimensional ultrasound was significantly better than conventional guidance in reducing the number of failed site cannulations from 6/17 (35 percent), to 0/12 (0 percent), p less than 0.05. Two-dimensional ultrasound also reduced the mean number of passes required to cannulate the vein from 3.12 to 1.75 (p less than .05), and was also successful in six/six (100) of patients who failed cannulation by conventional means (p less than 0.05). CONCLUSIONS: Intensivists can increase successful internal jugular vein cannulation using ultrasound guidance. Two-dimensional ultrasound should be considered for patients difficult to cannulate or those at high risk of cannulation complications.

Effects of ibuprofen and pentoxifylline on the cardiovascular response of normal humans to endotoxin.

Endotoxin is a major mediator of the life-threatening cardiovascular dysfunction that characterizes Gram-negative sepsis. In animal models of endotoxemia, pretreatment with ibuprofen or pentoxifylline attenuates some of these cardiovascular changes. To evaluate the effects of these agents on the human cardiovascular response to endotoxemia, hemodynamic variables were measured serially in 24 normal subjects who were given intravenous endotoxin. The subjects were randomized to receive oral ibuprofen (n = 9), pentoxifylline (n = 10), or no medication before endotoxin administration (n = 5). The subjects were volume loaded 3-5 h after endotoxin administration, and hemodynamic measurements were reassessed. Core temperature after endotoxin alone or endotoxin-pentoxifylline approached a maximum at 3 h (greater than or equal to 38.6 degrees C), while the endotoxin-ibuprofen group remained afebrile. At 3 and 5 h, all three groups had significant increases in heart rate, cardiac index, oxygen delivery, and oxygen consumption, while systemic vascular resistance index decreased significantly from baseline. The oxygen extraction ratio remained unchanged. After volume loading, the left ventricular ejection fraction and left ventricular end-diastolic and end-systolic volume indexes did not differ among the groups. The hyperdynamic cardiovascular response to endotoxin in humans occurs in the absence of fever and is not significantly ameliorated by oral cyclooxygenase or phosphodiesterase inhibition.

New perspectives on the management of septic shock in the cancer patient.

Septic shock is a common life-threatening problem, usually presenting with fever, tachycardia, tachypnea, and often a source of infection. The cardiac index is increased, with a decreased systemic vascular resistance, and a reversibly decreased ejection fraction with an increased end diastolic volume. The myocardial depression is most likely caused by a circulating humoral substance that depresses myocardial contractility. The initial treatment of septic shock is aggressive fluid resuscitation and antibiotic therapy, with vasopressors and inotropes being indicated in those patients who do not respond adequately to fluids. Therapy directed against the mediators of septic shock is theoretically promising, but to date has not been successful.

Site-directed mutagenesis of the genome of mouse hepatitis virus by targeted RNA recombination.

We have genetically characterized a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHV). This mutant, designated Alb4, is both temperature-sensitive and thermolabile, and its N protein is smaller than wild-type N. Sequence analysis of the Alb4 N gene revealed that it contains an internal deletion of 87 nucleotides, producing an in-frame deletion of 29 amino acids. All of these properties of Alb4 made it ideal for use as a recipient in a targeted RNA recombination experiment in which the deletion in Alb4 was repaired by recombination with synthetic RNA7, the smallest MHV subgenomic mRNA. Progeny from a cotransfection of Alb4 genomic RNA and synthetic RNA7 were selected for thermal stability. PCR analysis of candidate recombinants showed that they had regained the material that is deleted in the Alb4 mutant. They also had acquired a five nucleotide insertion in the 3' untranslated region, which had been incorporated into the synthetic RNA7 as a molecular tag. The presence of the tag was directly verified, as well, by sequencing the genomic RNA of purified recombinant viruses. This provided a clear genetic proof that the Alb4 phenotype was due to the observed deletion in the N gene. In addition, these results demonstrated that it is possible to obtain stable, independently replicating progeny from recombination between coronaviral genomic RNA and a tailored, synthetic RNA species. To date, we have constructed three additional mutants by this procedure. For two of these, a second-site point mutation that reverts the Alb4 phenotype has been transduced into a wild type background, which does not contain the Alb4 deletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiology, pathophysiology and clinical presentation of gram-negative sepsis.

OBJECTIVE: To review the epidemiology and pathophysiology of gram-negative sepsis and the new consensus terminology describing the clinical signs of sepsis. DATA SOURCES: Review of the medical literature and compiled data from animal and clinical trials. PARTICIPANTS: Members of the Society of Critical Care Medicine, American College of Chest Physicians and American Association of Critical-Care Nurses with expertise on the subject of sepsis and its complications. RESULTS: Preconference and general sessions were offered at the National Teaching Institutes of the American Association of Critical-Care Nurses, with the goal of clarifying the epidemiology, risk factors and pathophysiology of gram-negative sepsis. In addition, current terminology and new (1992) consensus terminology describing the clinical signs of sepsis were presented. Special emphasis was placed on the role of the healthcare provider in the prevention and recognition of sepsis and the role of the septic mediators in the septic cascade. CONCLUSIONS: If the incidence of sepsis is to be reduced, the healthcare provider must be aware of the risk factors for sepsis and methods of reducing nosocomial infections. A thorough understanding of the role of mediators and consensus terminology used to describe sepsis, severe sepsis, septic shock and multiple organ dysfunction syndrome is necessary to recognize early or progressive signs of sepsis and to initiate state-of-the-art therapy.

Pacemaker update 1984. Part III. Pacemaker arrhythmias in normally functioning pacemakers.

Familiarity with the more sophisticated modes of pacing is necessary to determine normal pacemaker function. A thorough knowledge of all functions of a pacemaker is required to interpret and manage pacemaker-induced arrhythmias.

Pacemaker update 1984. Part IV. DDD pacemaker--electrocardiographic assessment and problem solving.

Knowledge of the programmed parameters of an artificial pacemaker is critical to the accurate assessment of pacemaker function, especially with the use of an AV universal (DDD) pacemaker. In addition, each manufacturer's idiosyncrasies regarding pacemaker function have to be appreciated to intelligently monitor the rhythm of a state-of-the-art pacemaker.

Pacemaker update 1984. Part II. The pacemaker syndrome.

When ventricular pacemakers are implanted in patients who still retain normal AV conduction, retrograde conduction may activate the atria after ventricular activation. This results in a retrograde flow of blood from the ventricles to the atria with flooding of the jugular and pulmonary veins. Weakness or syncope can then occur as a result of lower cardiac output or congestive heart failure. A change to a dual-chamber pacemaker that produces AV synchrony is often necessary.