Monoclonal Antibodies against Accumulation-Associated Protein Affect EPS Biosynthesis and Enhance Bacterial Accumulation of Staphylococcus epidermidis

Because there is no effective antibiotic to eradicate Staphylococcus epidermidis biofilm infections that lead to the failure of medical device implantations, the development of anti-biofilm vaccines is necessary. Biofilm formation by S. epidermidis requires accumulation-associated protein (Aap) that contains sequence repeats known as G5 domains, which are responsible for the Zn2+-dependent dimerization of Aap to mediate intercellular adhesion. Antibodies against Aap have been reported to inhibit biofilm accumulation. In the present study, three monoclonal antibodies (MAbs) against the Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrpt1.5) were generated. MAb18B6 inhibited biofilm formation by S. epidermidis RP62A to 60% of the maximum, while MAb25C11 and MAb20B9 enhanced biofilm accumulation. All three MAbs aggregated the planktonic bacteria to form visible cell clusters. Epitope mapping revealed that the epitope of MAb18B6, which recognizes an identical area within AapBrpt constructs from S. epidermidis RP62A, was not shared by MAb25C11 and MAb20B9. Furthermore, all three MAbs were found to affect both Aap expression and extracellular polymeric substance (EPS, including extracellular DNA and PIA) biosynthesis in S. epidermidis and enhance the cell accumulation. These findings contribute to a better understanding of staphylococcal biofilm formation and will help to develop epitope-peptide vaccines against staphylococcal infections

Prp19 Arrests Cell Cycle via Cdc5L in Hepatocellular Carcinoma Cells

Pre-mRNA processing factor 19 (Prp19) is involved in many cellular events including pre-mRNA processing and DNA damage response. Recently, it has been identified as a candidate oncogene in hepatocellular carcinoma (HCC). However, the role of Prp19 in tumor biology is still elusive. Here, we reported that Prp19 arrested cell cycle in HCC cells via regulating G2/M transition. Mechanistic insights revealed that silencing Prp19 inhibited the expression of cell division cycle 5-like (Cdc5L) via repressing the translation of Cdc5L mRNA and facilitating lysosome-mediated degradation of Cdc5L in HCC cells. Furthermore, we found that silencing Prp19 induced cell cycle arrest could be partially resumed by overexpressing Cdc5L. This work implied that Prp19 participated in mitotic progression and thus could be a promising therapeutic target of HCC

Blood pressure and left ventricular function changes in different ambulatory blood pressure patterns at high altitude

Acute high‐altitude (HA) exposure induces physiological responses of the heart and blood pressure (BP). However, few studies have investigated the responses associated with dipper and non‐dipper BP patterns. In this prospective study, 72 patients underwent echocardiography and 24‐h ambulatory BP testing at sea level and HA. Patients were divided into dipper and non‐dipper groups according to BP at sea level. Acute HA exposure elevated 24‐h systolic and diastolic BP and increased BP variability, particularly in the morning. Moreover, acute exposure increased left ventricular torsion, end‐systolic elastance, effective arterial elastance, and untwisting rate, but reduced peak early diastolic velocity/late diastolic velocity and peak early diastolic velocity/early diastolic velocity, implying enhanced left ventricular systolic function but impaired filling. Dippers showed pronounced increases in night‐time BP, while non‐dippers showed significant elevation in day‐time BP, which blunted differences in nocturnal BP fall, and lowest night‐time and evening BP. Dippers had higher global longitudinal strain, torsion, and untwisting rates after acute HA exposure. Variations in night‐time systolic BP correlated with variations in torsion and global longitudinal strain. Our study firstly demonstrates BP and cardiac function variations during acute HA exposure in different BP patterns and BP increases in dippers at night, while non‐dippers showed day‐time increases. Furthermore, enhanced left ventricular torsion and global longitudinal strain are associated with BP changes. Non‐dippers showed poor cardiac compensatory and maladaptive to acute HA exposure. However, the exact mechanisms involved need further illumination

Prognostic significance of day‐by‐day in‐hospital blood pressure variability in COVID‐19 patients with hypertension

Abstract Hypertension is the most common comorbidity in patients with coronavirus disease 2019 (COVID‐19) and increases in‐hospital mortality. Day‐by‐day blood pressure (BP) variability (BPV) is associated with clinical outcomes in hypertensive patients. However, little information is available on the association of BPV with the outcomes of COVID‐19 patients with hypertension. This study aimed to demonstrate whether day‐by‐day in‐hospital BPV had prognostic significance in these patients. The authors included 702 COVID‐19 patients with hypertension from Huoshenshan Hospital (Wuhan, China), who underwent valid in‐hospital BP measurements on at least seven consecutive days. Day‐by‐day BPV was assessed by standard deviation (SD), coefficient of variation (CV), and variation independent of mean (VIM). Overall, patients with severe COVID‐19 and non‐survivors had higher BPV than moderate cases and survivors, respectively. Additionally, higher BPV was correlated with greater age and higher levels of C‐reactive protein, procalcitonin, high‐sensitive cardiac troponin I, and B‐type natriuretic peptide. In multivariable Cox regression, SD of systolic BP (SBP) was predictive of mortality [hazard ratio (HR) 1.17, 95% confidence interval (CI) 1.05–1.30] as well as acute respiratory distress syndrome (ARDS) (HR 1.09, 95% CI 1.01–1.16). Similar trends were observed for CV and VIM of SBP, but not indices of diastolic BP variability. The authors demonstrated that day‐by‐day in‐hospital SBP variability can independently predict mortality and ARDS in COVID‐19 patients with hypertension. And high BPV might be correlated with severe inflammation and myocardial injury. Further studies are needed to clarify whether early reduction of BPV will improve the prognosis of these patients

Aap expression in <i>S. epidermidis</i>.

<p>(A) Protein expression level. Aap expression in <i>S. epidermidis</i> RP62A co-cultured with each MAb (10 µg/mL) was detected using Western blot with MAb_25C11 (1 ng/mL). After separation of the proteins using 7% SDS-PAGE, the gel pieces carrying high molecular-weight proteins (>130 kDa) were excised for Western blot assay, and the remaining gel was stained using Coomassie brilliant blue as the endogenous control. (B) Relative transcriptional level. The transcriptional level of <i>aap</i> was detected by Q-RT-PCR using RNA sample extracted from <i>S. epidermidis</i> RP62A co-cultured with each MAb (10 µg/mL). The housekeeping gene <i>gyrB</i> was used as an endogenous control, and all samples were analyzed in triplicate and normalized against <i>gyrB</i> transcription (means ± SD). “RP62A”: untreated, “Mock”: normal mouse IgG-treated.</p

Oligonucleotide primers used in the present study.

<p><b>a</b>. Primers were designed according to the genomic sequence of <i>S. epidermidis</i> ATCC 12228 (GenBank NC_004461).</p><p><b>b</b>. Primers were designed according to the genomic sequence of <i>S. epidermidis</i> RP62A (GenBank NC_002976).</p><p><b>c</b>. Primers were designed according to the gene sequence of the 56-residue B1 immunoglobulin binding domain (GB1) of immunoglobulin G-binding protein from <i>Streptococcus dysgalactiae</i> subsp. <i>equisimilis</i> GGS_124 (amino acids 303–357, GenBank YP_002997067).</p

Epitope mapping of anti-AapBrpt1.5 MAbs.

<p>AapBrpt1.5 N-terminally fused with a GB1-tagged six-histidine (GB1-His) tag was truncated into a series of fragments as shown in the schematic diagrams, and the binding ability between the truncated fragments and MAbs was analyzed using immunoprecipitation. (A) Preliminary epitope mapping. (B) Precise recognition site mapping of MAb_25C11 and MAb_20B9. (C) Precise recognition site mapping of MAb_18B6. (D) Domain structures of Aap from <i>S. epidermidis</i> RP62A (“RP62A”) and ATCC 12228 (“12228”). The A-repeat region, the putative globular domain (α/β), the B-repeat region containing 6 or 12 tandem Brpt constructs, the collagen-like proline/glycine-rich region, the domain boundary of AapBrpt1.5, and the MAb epitopes are illustrated. (E) Amino acid sequence alignment of AapBrpt constructs. The AapBrpt construct in AapBrpt1.5 (GenBank NP_763730) and twelve distinct AapBrpt constructs from <i>S. epidermidis</i> RP62A (RP62A 01-12, GenBank YP_189945) were aligned using the ClustalW2 program (<a href="http://www.ebi.ac.uk/Tools/clustalw2" target="_blank">http://www.ebi.ac.uk/Tools/clustalw2</a>). The identified epitopes of the MAbs are shown in boxes, and the identical residues are marked with asterisks. The conserved substitutions are represented by “:”, and semi-conserved substitutions are represented by “.”. Two conserved His residues in AapBrpt constructs are marked with triangles.</p

Aap expression in biofilms of <i>S. epidermidis</i>.

<p>Aap in the biofilms of <i>S. epidermidis</i> RP62A was probed with MAb_25C11 (10 ng/mL) and Cy3-conjugated secondary antibody (1∶100 diluted, red fluorescence), and the bacteria were further stained with SYTO9 (1 µM, green fluorescence). Aap expression was observed under a Leica TCS SP5 CLSM. Confocal microscopy Z-series of the biofilms were acquired in 0.5-µm increments. “PC”: positive control (antigens contained in the biofilm were probed using mouse anti-<i>S. epidermidis</i> serum (1∶400 diluted) and Cy3-conjugated secondary antibody, showing that antibodies could diffuse to the inner side of the biofilm), “NC”: negative control (the biofilm formed in the presence of MAb_25C11 was probed with Cy3-conjugated secondary antibody alone to establish that the MAb-treated biofilms no longer contained initially added MAb (10 µg/mL), that could cause false-positive immunofluorescence, after 14 h culture), “RP62A”: untreated, “Mock”: normal mouse IgG-treated, the red arrow indicates the crater-like micropores.</p

Cell aggregation mediated by the MAbs.

<p><i>S. epidermidis</i> RP62A was statically cultured in TSB medium containing each MAb (10 µg/mL, 0.07 µM) alone or both MAb (10 µg/mL, 0.07 µM) and AapBrpt1.5 (untagged, 3.2 µg/mL, 0.14 µM). The photomicrographs were obtained by a Nikon TE2000-U inverted microscopy using a 40x objective lens (Nikon, <a href="http://www.nikoninstruments.com/" target="_blank">http://www.nikoninstruments.com/</a>). “RP62A”: untreated; “Mock”: normal mouse IgG-treated.</p