Cardiac Protein Kinase D1 ablation alters the myocytes ß-adrenergic response.

ß-adrenergic (ß-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute ß-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte ß-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal ß-adrenergic responses in Ca2+ handling.

Systematic review and meta-analysis of Veress needle entry versus direct trocar entry in gynecologic surgery.

Objective: Although many studies have been performed, no consensus exists as to the ideal entry for laparoscopic gynecologic surgery. We sought out to compare the safety of direct trocar insertion with that of the Veress needle entry technique in gynecologic laparoscopic surgery. Design: Systematic review with meta-analysis. Setting: We searched Medline, ClinicalTrials.Gov, PubMed, Cochrane CENTRAL, SCOPUS, and Web of Science from their inception through 31 July 2021 for relevant studies. We included only controlled trials and ultimately seven trials were included in our meta-analysis. Participants: Inclusion criteria included women undergoing gynecological laparoscopic surgery. Intervention: The intervention of direct trocar insertion technique compared with Veress needle entry technique. Main outcome measures: We compared five different outcomes associated with the efficacy and complications of laparoscopic entry. Results: The pooled analysis showed that Veress needle entry was associated with a significant increase in the incidences of extraperitoneal insufflation (RR=0.177, 95% Cl (0.094 to 0.333), p<0.001), omental injury (RR=0.418, 95% Cl (0.195 to 0.896), p<0.001), failed entry (RR=0.173, 95% Cl (0.102 to 0.292), p<0.001), and trocar site infection (RR=0.404, 95% Cl (0.180 to 0.909), p<0.029). There was no significant difference between the two groups regarding the visceral injury (RR=0.562, 95% Cl (0.047 to 6.676), p<0.648). Conclusions: When excluding all data apart from gynecologic surgery, the Veress needle entry technique may have an increased incidence of some, but not all complications of laparoscopic entry. It may also have a higher incidence of failed entry compared with direct entry techniques. Care should be taken in extrapolating these general results to specific surgeon experience levels. Trial registration number: CRD42021273726.

Contained and uncontained morcellation in hysterectomy and myomectomy: A systematic review and meta-analysis

We sought to analyze all high-quality studies available regarding the possible differences in contained and uncontained techniques for morcellation of fibroids and uteri. We systematically searched PubMed, Cochrane Central, Scopus, ClinicalTrials.Gov, MEDLINE and Web of Science from September 2010 to September 2020 for our search terms. We included studies that specifically enrolled patients undergoing power morcellation myomectomy or power morcellation hysterectomy procedures. In our search, we had no restriction to age, country, or publication date. We extracted data related to study design, baseline characteristics of patients, and perioperative outcomes such as total operative time, total blood loss, and duration of hospital stay. We found no substantial difference in total operative time between contained power morcellation and uncontained manual morcellation myomectomy (p=0.52), but contained power morcellation had a significantly longer total operative time than uncontained power morcellation for hysterectomy and myomectomy [135.50 vs. 93.33 minutes, (p=0.003)]. Total blood loss was comparable for contained power morcellation versus uncontained manual morcellation myomectomy (p=0.32) and contained power morcellation versus uncontained power morcellation myomectomy or hysterectomy (p=0.91). Contained power morcellation and uncontained manual morcellation myomectomy had comparable hospital stay periods (p=0.5). Contained power morcellation leads to a longer operating time than uncontained power morcellation for both hysterectomy and myomectomy. No differences were found in comparisons of blood loss, operative time, or comparison to manual methods of morcellation.

Effect of single- and double-layer cesarean section closure on residual myometrial thickness and isthmocele - a systematic review and meta-analysis

Objective: To determine the incidence of isthmocele, its effect on residual myometrial thickness (RMT), and other complications of Cesarean delivery (CD) in relation to single- and double-layer CD closure. We searched PubMed, SCOPUS, Web of Science, ClinicalTrials.gov, MEDLINE and Cochrane Library for relevant clinical trials assessing the use of single- and double-layer uterine closure in patients undergoing cesarean sections from inception through to March 2021. Materials and Methods: Our population was women undergoing cesarean section with uterine closure by any double-layer method, compared with those undergoing uterine closure through a single-layer method. RMT (in mm) was measured at 6 weeks, niche/isthmocele existence at 6 weeks, RMT (in mm) at 6-24 months and niche/isthmocele existence at 6-24 months. In order to present the highest quality evidence, we only included clinical trials in our analysis. To perform this review, we reported dichotomous outcomes using percent and total, while continuous outcomes were reported using mean ± standard deviations, and relative 95% confidence intervals using the inverse variance method. Results: We found that the RMT in the double-layer closure group was significantly higher at six weeks [mean difference (MD)=-0.43 (-0.77, -0.09)], (p=0.01) and at 6-24 months of follow-up [MD=-1.27 (-2.28, -0.25)], (p=0.01). The incidence of isthmocele in the two groups, as well as the other investigated outcomes were similar across the different groups. Conclusion: High-quality evidence shows that double-layer closure results in a higher RMT compared with a single-layer closure, despite no significant difference in isthmocele formation.

The Beta Cell in Type 2 Diabetes.

PURPOSE OF REVIEW: This review summarizes the alterations in the ß-cell observed in type 2 diabetes (T2D), focusing on changes in ß-cell identity and mass and changes associated with metabolism and intracellular signaling. RECENT FINDINGS: In the setting of T2D, ß-cells undergo changes in gene expression, reverting to a more immature state and in some cases transdifferentiating into other islet cell types. Alleviation of metabolic stress, ER stress, and maladaptive prostaglandin signaling could improve ß-cell function and survival. The ß-cell defects leading to T2D likely differ in different individuals and include variations in ß-cell mass, development, ß-cell expansion, responses to ER and oxidative stress, insulin production and secretion, and intracellular signaling pathways. The recent recognition that some ß-cells undergo dedifferentiation without dying in T2D suggests strategies to revive these cells and rejuvenate their functionality.

Combination of anthracyclines and anti-CD47 therapy inhibit invasive breast cancer growth while preventing cardiac toxicity by regulation of autophagy.

BACKGROUND: A perennial challenge in systemic cytotoxic cancer therapy is to eradicate primary tumors and metastatic disease while sparing normal tissue from off-target effects of chemotherapy. Anthracyclines such as doxorubicin are effective chemotherapeutic agents for which dosing is limited by development of cardiotoxicity. Our published evidence shows that targeting CD47 enhances radiation-induced growth delay of tumors while remarkably protecting soft tissues. The protection of cell viability observed with CD47 is mediated autonomously by activation of protective autophagy. However, whether CD47 protects cancer cells from cytotoxic chemotherapy is unknown. METHODS: We tested the effect of CD47 blockade on cancer cell survival using a 2-dimensional high-throughput cell proliferation assay in 4T1 breast cancer cell lines. To evaluate blockade of CD47 in combination with chemotherapy in vivo, we employed the 4T1 breast cancer model and examined tumor and cardiac tissue viability as well as autophagic flux. RESULTS: Our high-throughput screen revealed that blockade of CD47 does not interfere with the cytotoxic activity of anthracyclines against 4T1 breast cancer cells. Targeting CD47 enhanced the effect of doxorubicin chemotherapy in vivo by reducing tumor growth and metastatic spread by activation of an anti-tumor innate immune response. Moreover, systemic suppression of CD47 protected cardiac tissue viability and function in mice treated with doxorubicin. CONCLUSIONS: Our experiments indicate that the protective effects observed with CD47 blockade are mediated through upregulation of autophagic flux. However, the absence of CD47 in did not elicit a protective effect in cancer cells, but it enhanced macrophage-mediated cancer cell cytolysis. Therefore, the differential responses observed with CD47 blockade are due to autonomous activation of protective autophagy in normal tissue and enhancement immune cytotoxicity against cancer cells.

Molecular characterization of patient-derived human pancreatic tumor xenograft models for preclinical and translational development of cancer therapeutics.

Preclinical evaluation of novel cancer agents requires models that accurately reflect the biology and molecular characteristics of human tumors. Molecular profiles of eight pancreatic ductal adenocarcinoma patient tumors were compared to corresponding passages of xenografts obtained by grafting tumor fragments into immunocompromised mice. Molecular characterization was performed by copy number analysis, gene expression and microRNA microarrays, mutation analysis, short tandem repeat (STR) profiling, and immunohistochemistry. Xenografts were found to be highly representative of their respective tumors, with a high degree of genetic stability observed by STR profiling and mutation analysis. Copy number variation (CNV) profiles of early and late xenograft passages were similar, with recurrent losses on chromosomes 1p, 3p, 4q, 6, 8p, 9, 10, 11q, 12p, 15q, 17, 18, 20p, and 21 and gains on 1q, 5p, 8q, 11q, 12q, 13q, 19q, and 20q. Pearson correlations of gene expression profiles of tumors and xenograft passages were above 0.88 for all models. Gene expression patterns between early and late passage xenografts were highly stable for each individual model. Changes observed in xenograft passages largely corresponded to human stromal compartment genes and inflammatory processes. While some differences exist between the primary tumors and corresponding xenografts, the molecular profiles remain stable after extensive passaging. Evidence for stability in molecular characteristics after several rounds of passaging lends confidence to clinical relevance and allows for expansion of models to generate the requisite number of animals required for cohorts used in drug screening and development studies.

Safety of prophylactic anticoagulation at a pediatric hospital.

The importance of preventing venous thromboembolism (VTE) in hospitalized adults is well recognized. We recently developed and published our institutional guidelines for the prevention of VTE in high-risk hospitalized patients in a pediatric hospital. The objective of this prospective observational study was to evaluate the safety of anticoagulation after these guidelines were instituted. The primary outcome was major bleeding and secondary outcomes included minor bleeding and VTE. Eighty-nine patients were enrolled, with a mean age of 16.6 years. The most common risk factors for VTE were impaired mobility, lower extremity orthopedic surgery, and obesity. The majority of patients (63%) had 3 or more risk factors. There were 2 major bleeding events, and minor bleeding occurred in 5 patients, all in patients who had undergone major orthopedic surgery. Therefore the risk of major bleeding in orthopedic surgery patients was 4% (2/51), and 0% (0/38) in the remaining patients. No patient developed a non-catheter-related VTE, which was the primary intent of our guidelines. Although there remains much work to be done to optimize VTE strategies in pediatric patients, this study provides information regarding the risks of VTE prophylaxis using a pragmatic approach in hospitalized patients with multiple risk factors for VTE. More studies are needed to better define the risk:benefit ratio in this population.

Duplication and diversification of the LEAFY HULL STERILE1 and Oryza sativa MADS5 SEPALLATA lineages in graminoid Poales.

BACKGROUND: Gene duplication and the subsequent divergence in function of the resulting paralogs via subfunctionalization and/or neofunctionalization is hypothesized to have played a major role in the evolution of plant form. The LEAFY HULL STERILE1 (LHS1) SEPALLATA (SEP) genes have been linked with the origin and diversification of the grass spikelet, but it is uncertain 1) when the duplication event that produced the LHS1 clade and its paralogous lineage Oryza sativa MADS5 (OSM5) occurred, and 2) how changes in gene structure and/or expression might have contributed to subfunctionalization and/or neofunctionalization in the two lineages. METHODS: Phylogenetic relationships among 84 SEP genes were estimated using Bayesian methods. RNA expression patterns were inferred using in situ hybridization. The patterns of protein sequence and RNA expression evolution were reconstructed using maximum parsimony (MP) and maximum likelihood (ML) methods, respectively. RESULTS: Phylogenetic analyses mapped the LHS1/OSM5 duplication event to the base of the grass family. MP character reconstructions estimated a change from cytosine to thymine in the first codon position of the first amino acid after the Zea mays MADS3 (ZMM3) domain converted a glutamine to a stop codon in the OSM5 ancestor following the LHS1/OSM5 duplication event. RNA expression analyses of OSM5 co-orthologs in Avena sativa, Chasmanthium latifolium, Hordeum vulgare, Pennisetum glaucum, and Sorghum bicolor followed by ML reconstructions of these data and previously published analyses estimated a complex pattern of gain and loss of LHS1 and OSM5 expression in different floral organs and different flowers within the spikelet or inflorescence. CONCLUSIONS: Previous authors have reported that rice OSM5 and LHS1 proteins have different interaction partners indicating that the truncation of OSM5 following the LHS1/OSM5 duplication event has resulted in both partitioned and potentially novel gene functions. The complex pattern of OSM5 and LHS1 expression evolution is not consistent with a simple subfunctionalization model following the gene duplication event, but there is evidence of recent partitioning of OSM5 and LHS1 expression within different floral organs of A. sativa, C. latifolium, P. glaucum and S. bicolor, and between the upper and lower florets of the two-flowered maize spikelet.

Astrocytes from familial and sporadic ALS patients are toxic to motor neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for <2% of all ALS cases. Their role in sporadic (S)ALS, which affects >90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non-cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.

B- and C-class gene expression during corona development of the blue passionflower (Passiflora caerulea, Passifloraceae).

PREMISE OF STUDY: The origin of the passionflower corona, a complex series of structures between the petals and stamens, has intrigued botanists for centuries, but has proven intractable using traditional approaches. Supplementing developmental data with expression analyses of three floral identity genes, we test whether the corona in Passiflora caerulea (blue passionflower) is homologous to petals or stamens or whether an alternative hypothesis of the corona as a novel structure is supported. METHODS: Corona development was investigated using scanning electron microscopy. Expression of the P. caerulea B-class genes PISTILLATA (PcPI) and TOMATO MADS6 (PcTM6), and C-class gene AGAMOUS (PcAG) was investigated using a combination of RT-PCR and mRNA in situ hybridization analyses. KEY RESULTS: Corona development starts as a ring of tissue at the base of petals. The outer radii and operculum initiate first at the periphery, followed by the inner radii and pali toward the center, and finally an annulus beneath the operculum. Late in development, a limen, the innermost component of the corona, develops from the side of the androgynophore. RT-PCR analyses indicate that the B-class genes PcPI and PcTM6 and C-class gene PcAG were all expressed in mature coronas. However, mRNA in situ hybridization analyses revealed complex temporal patterns of gene expression in the different corona elements. CONCLUSIONS: Our data support the hypothesis that the corona is a composite structure, with the radii, pali, and operculum homologous to stamens, and the limen, which only expresses PcTM6, considered to be a novel structure distinct from the androgynophore.

vanishing tassel2 encodes a grass-specific tryptophan aminotransferase required for vegetative and reproductive development in maize.

Auxin plays a fundamental role in organogenesis in plants. Multiple pathways for auxin biosynthesis have been proposed, but none of the predicted pathways are completely understood. Here, we report the positional cloning and characterization of the vanishing tassel2 (vt2) gene of maize (Zea mays). Phylogenetic analyses indicate that vt2 is a co-ortholog of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1), which converts Trp to indole-3-pyruvic acid in one of four hypothesized Trp-dependent auxin biosynthesis pathways. Unlike single mutations in TAA1, which cause subtle morphological phenotypes in Arabidopsis thaliana, vt2 mutants have dramatic effects on vegetative and reproductive development. vt2 mutants share many similarities with sparse inflorescence1 (spi1) mutants in maize. spi1 is proposed to encode an enzyme in the tryptamine pathway for Trp-dependent auxin biosynthesis, although this biochemical activity has recently been questioned. Surprisingly, spi1 vt2 double mutants had only a slightly more severe phenotype than vt2 single mutants. Furthermore, both spi1 and vt2 single mutants exhibited a reduction in free auxin levels, but the spi1 vt2 double mutants did not have a further reduction compared with vt2 single mutants. Therefore, both spi1 and vt2 function in auxin biosynthesis in maize, possibly in the same pathway rather than independently as previously proposed.

MADS-box gene expression and implications for developmental origins of the grass spikelet.

Basic questions regarding the origin and evolution of grass (Poaceae) inflorescence morphology remain unresolved, including the developmental genetic basis for evolution of the highly derived outer spikelet organs. To evaluate homologies between the outer sterile organs of grass spikelets and inflorescence structures of nongrass monocot flowers, we describe expression patterns of APETALA1/FRUITFULL-like (AP1/FUL) and LEAFY HULL STERILE-like (LHS1) MADS-box genes in an early-diverging grass (Streptochaeta angustifolia) and a nongrass outgroup (Joinvillea ascendens). AP1/FUL-like genes are expressed only in floral organs of J. ascendens, supporting the hypothesis that they mark the floral boundary in nongrass monocots, and JaLHS1/OsMADS5 is expressed in the inner and outer tepals, stamen filaments and pistil. In S. angustifolia, SaFUL2 is expressed in all 11 (or 12) bracts of the primary inflorescence branch, but not in the suppressed floral bract below the abscission zone. In contrast, SaLHS1 is only expressed in bracts 6-11 (or 12). Together, these data are consistent with the hypotheses that (1) bracts 1-5 of S. angustifolia primary inflorescence branches and glumes of grass spikelets are homologous and that (2) the outer tepals of immediate grass relatives, bracts 6-8 of S. angustifolia, and the lemma/palea are homologous, although other explanations are possible.