A cell abundance analysis based on efficient PAM clustering for a better understanding of the dynamics of endometrial remodelling.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) is a powerful tool for investigating cell abundance changes during tissue regeneration and remodeling processes. Differential cell abundance supports the initial clustering of all cells; then, the number of cells per cluster and sample are evaluated, and the dependence of these counts concerning the phenotypic covariates of the samples is studied. Analysis heavily depends on the clustering method. Partitioning Around Medoids (PAM or k-medoids) represents a well-established clustering procedure that leverages the downstream interpretation of clusters by pinpointing real individuals in the dataset as cluster centers (medoids) without reducing dimensions. Of note, PAM suffers from high computational costs and memory requirements. RESULTS: This paper proposes a method for differential abundance analysis using PAM as a clustering method and negative binomial regression as a statistical model to relate covariates to cluster/cell counts. We used this approach to study the differential cell abundance of human endometrial cell types throughout the natural secretory phase of the menstrual cycle. We developed a new R package -scellpam-, that incorporates an efficient parallel C++ implementation of PAM, and applied this package in this study. We compared the PAM-BS clustering method with other methods and evaluated both the computational aspects of its implementation and the quality of the classifications obtained using distinct published datasets with known subpopulations that demonstrate promising results. CONCLUSIONS: The implementation of PAM-BS, included in the scellpam package, exhibits robust performance in terms of speed and memory usage compared to other related methods. PAM allowed quick and robust clustering of sets of cells with a size ranging from 70,000 to 300,000 cells. https://cran.r-project.org/web/packages/scellpam/index.html . Finally, our approach provides important new insights into the transient subpopulations associated with the fertile time frame when applied to the study of changes in the human endometrium during the secretory phase of the menstrual cycle.

The human periconceptional maternal-embryonic space in health and disease.

Pregnancy is established during the periconceptional period as a continuum beginning with blastocyst attachment to the endometrial epithelial surface followed by embryo invasion and placenta formation. This period sets the foundation for the child and mother's health during pregnancy. Emerging evidence indicates that prevention of downstream pathologies in both the embryo/newborn and pregnant mother may be possible at this stage. In this review, we discuss current advances in the periconceptional space, including the preimplantation human embryo and maternal endometrium. We also discuss the role of the maternal decidua, the periconceptional maternal-embryonic interface, the dialogue between these elements, and the importance of the endometrial microbiome in the implantation process and pregnancy. Finally, we discuss the myometrium in the periconceptional space and review its role in determining pregnancy health.

Aberrant Alternative Splicing in U2af1/Tet2 Double Mutant Mice Contributes to Major Hematological Phenotypes.

Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3' splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1mut/+), studied the U2af1mut/+ hematopoietic system, and did not observe any gross differences in both young (12-13 weeks) and old (23 months) U2af1mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1mut/+Tet2-/- mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1mut/+Tet2-/- mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.

Cost-minimization analysis favors outpatient quick diagnosis unit over hospitalization for the diagnosis of potentially serious diseases.

BACKGROUND: Quick diagnosis units (QDUs) are a promising alternative to conventional hospitalization for the diagnosis of suspected serious diseases, most commonly cancer and severe anemia. Although QDUs are as effective as hospitalization in reaching a timely diagnosis, a full economic evaluation comparing both approaches has not been reported. AIMS: To evaluate the costs of QDU vs. conventional hospitalization for the diagnosis of cancer and anemia using a cost-minimization analysis on the proven assumption that health outcomes of both approaches were equivalent. METHODS: Patients referred to the QDU of Bellvitge University Hospital of Barcelona over 51 months with a final diagnosis of severe anemia (unrelated to malignancy), lymphoma, and lung cancer were compared with patients hospitalized for workup with the same diagnoses. The total cost per patient until diagnosis was analyzed. Direct and non-direct costs of QDU and hospitalization were compared. RESULTS: Time to diagnosis in QDU patients (n=195) and length-of-stay in hospitalized patients (n=237) were equivalent. There were considerable costs savings from hospitalization. Highest savings for the three groups were related to fixed direct costs of hospital stays (66% of total savings). Savings related to fixed non-direct costs of structural and general functioning were 33% of total savings. Savings related to variable direct costs of investigations were 1% of total savings. Overall savings from hospitalization of all patients were €867,719.31. CONCLUSION: QDUs appear to be a cost-effective resource for avoiding unnecessary hospitalization in patients with anemia and cancer. Internists, hospital executives, and healthcare authorities should consider establishing this model elsewhere.

Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients.

Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.

A quick diagnosis unit as an alternative to conventional hospitalization in a tertiary public hospital: a descriptive study.

INTRODUCTION:  Reports indicate that a significant number of patients admitted to internal medicine units could be studied on an outpatient basis. OBJECTIVES:  This article assesses a quick diagnosis unit (QDU) as an alternative to acute hospitalization for the diagnostic study of patients with potentially serious diseases and suspected malignancy.  PATIENTS AND METHODS:  Between March 2008 and June 2012, 1226 patients were attended by the QDU. Patients were referred from the emergency department, primary health care centers, and outpatient clinics according to well­defined criteria. Clinical information was prospectively registered in a database.  RESULTS:  There were 634 men (51.7%), with a mean age of 60.5 ±17.5 years. The mean time to the first visit was 3.5 ±5.3 days. Most patients (65.7%) required only 2 visits. The mean interval to diagnosis was 12.2 ±14.7 days. A total of 324 patients (26.4%) had cancer. The diagnosis was  solid tumor in 81.5% of the cases, lymphoma in 19.8%, and various hematologic malignancies in 4.3%. The second most common diagnosis was anemia not associated with cancer (8.6% of the cases). Admission to the QDU allowed to avoid conventional hospitalization for diagnostic studies in 71.5% of the patients, representing a mean freeing­up rate of 7 internal medicine beds per day. In a satisfaction survey, 97% of the patients were completely or very satisfied and 96% preferred the QDU to conventional hospitalization.  CONCLUSIONS:  A QDU may be a feasible alternative to conventional hospitalization for the diagnosis of otherwise healthy patients with suspected severe disease. Appropriately managed and supported, QDUs can lighten the burden of emergency departments and reduce the need for hospitals beds.

Impaired expression of DICER, DROSHA, SBDS and some microRNAs in mesenchymal stromal cells from myelodysplastic syndrome patients.

UNLABELLED: Background Recent findings suggest that a specific deletion of Dicer1 in mesenchymal stromal cell-derived osteoprogenitors triggers several features of myelodysplastic syndrome in a murine model. Our aim was to analyze DICER1 and DROSHA gene and protein expression in mesenchymal stromal cells (the osteoblastic progenitors) obtained from bone marrow of myelodysplastic syndrome patients, in addition to microRNA expression profile and other target genes such as SBDS, a DICER1-related gene that promotes bone marrow dysfunction and myelodysplasia when repressed in a murine model. DESIGN AND METHODS: Mesenchymal stromal cells from 33 bone marrow samples were evaluated. DICER, DROSHA and SBDS gene expression levels were assessed by real-time PCR and protein expression by Western blot. MicroRNA expresion profile was analyzed by commercial low-density arrays and some of these results were confirmed by individual real-time PCR. RESULTS: Mesenchymal stromal cells from myelodysplastic syndrome patients showed lower DICER1 (0.65±0.08 vs. 1.91±0.57; P=0.011) and DROSHA (0.62±0.06 vs. 1.38±0.29; P=0.009) gene expression levels, two relevant endonucleases associated to microRNA biogenesis, in comparison to normal myelodysplastic syndrome. These findings were confirmed at protein levels by Western blot. Strikingly, no differences were observed between paired mononuclear cells from myelodysplastic syndrome and controls. In addition, mesenchymal stromal cells from myelodysplastic syndrome patients showed significant lower SBDS (0.63±0.06 vs. 1.15±0.28; P=0.021) gene expression levels than mesenchymal stromal cells from healthy controls. Furthermore, mesenchymal stromal cells from myelodysplastic syndrome patients showed an underlying microRNA repression compared to healthy controls. Real-time PCR approach confirmed that mir-155, miR-181a and miR-222 were down-expressed in mesenchymal stromal cells from myelodysplastic syndrome patients. Conclusions This is the first description of an impaired microRNA biogenesis in human mesenchymal stromal cells from myelodysplastic syndrome patients, where DICER1 and DROSHA gene and protein downregulation correlated to a gene and microRNA abnormal expression profile, validating the animal model results previously described.

Prevalence and routine assessment of unhealthy alcohol use in hospitalized patients.

OBJECTIVES: To determine the prevalence of alcohol misuse among medical inpatients and the methods used by medical staff to evaluate alcohol consumption. METHODS: Multicenter, prospective, observational, cross-sectional study performed at 21 hospitals in Spain. All adult patients hospitalized in internal medicine wards on 12 March 2008 were eligible for study. Alcohol consumption was evaluated with the Alcohol Use Disorders Identification Test (AUDIT-C and AUDIT) and the Systematic Inventory of Alcohol Consumption questionnaire. Drinking patterns were determined according to clinical evaluation using ICD-10 criteria. Medical records were reviewed to gather information on the recording of alcohol use. RESULTS: We assessed 1039 inpatients, of whom 123 (12%) had unhealthy alcohol drinking patterns. Alcohol misuse was more frequent among males (odds ratio 5.20), younger patients (odds ratio, 14.17), median age patients (odds ratio, 2.99), and South Region (odds ratio, 1.77). Alcohol use during hospitalization was recorded in 603 inpatients (59%); quantitative records were performed in 28% of hazardous and harmful drinkers and in 41% of dependent patients. Lack of alcohol use recording was more frequent among females (odds ratio 1.73), median and older age groups (odds ratios 1.44 and 1.73, respectively), Northwest Regions (odds ratios 3.46). Patients from the East Region (odds ratio 0.47) had more frequently assessed the question in their medical records. CONCLUSIONS: Prevalence of alcohol misuse was higher in hospitalized patients than in the general population. Adequate quantitative recording was infrequent. We stress the need to implement measures to increase and improve the detection and recording of alcohol use.

Community-acquired pneumonia in very elderly patients: causative organisms, clinical characteristics, and outcomes.

We performed an observational analysis of prospectively collected data on 1,474 adult patients who were hospitalized for community-acquired pneumonia; 1,169 patients were under 80 years of age and 305 (21%) patients were over 80 years ("very elderly"). Mean patient ages were 60 years in the former group and 85 years in the latter group. Severely immunosuppressed patients and nursing-home residents were not included. Comorbidities significantly associated with older age were chronic obstructive pulmonary disease, chronic heart disease, and dementia. The most common causative organism was Streptococcus pneumoniae (23% in both groups). Aspiration pneumonia was more frequent in the very elderly (5% in younger patients versus 10% in the very elderly); Legionella pneumophila (8% in younger patients versus 1% in the very elderly) and atypical agents (7% in younger patients versus 1% in the very elderly) were rarely recorded in the very elderly. While very elderly patients complained less frequently of pleuritic chest pain, headache, and myalgias, they were more likely to have absence of fever and altered mental status on admission. No significant differences were observed between groups as regards incidence of classic bacterial pneumonia syndrome (60% versus 59%) in 343 patients with pneumococcal pneumonia. The development of inhospital complications (26% in younger versus 32% in very elderly patients) as well as early mortality (2% in younger versus 7% in very elderly patients) and overall mortality (6% in younger versus 15% very elderly patients) were significantly higher in very elderly patients. Acute respiratory failure and shock/multiorgan failure were the most frequent causes of death, especially of early mortality. Factors independently associated with 30-day mortality in the very elderly were altered mental status on admission (odds ratio, 3.69), shock (odds ratio, 10.69), respiratory failure (odds ratio, 3.50), renal insufficiency (odds ratio, 5.83), and Gram-negative pneumonia (odds ratio, 20.27).

Resistance mutations in HIV-infected patients experiencing early failure with nelfinavir-containing triple combinations.

BACKGROUND: The purpose of our study was to assess the presence of nelfinavir (NFV)-associated resistance mutations at the time of early virological failure in subjects receiving NFV as part of a first protease inhibitor (PI)-based triple regimen. MATERIAL/METHODS: Subjects failing their first PI-based NFV-containing triple regimen were identified in six Spanish hospitals. HIV genotyping was carried out in plasma samples collected at the time of the first viral rebound. RESULTS: Upon initiation of NFV-based therapy, 19 of the 30 subjects (63%) were naïve; 11 (37%) had been exposed to nucleoside analogues. Median HIV-RNA at the time of viral rebound was 4, 180 copies/ml. PCR-amplified products were obtained in 22 subjects (73%). These products were sequenced and primary PI resistance mutations were recognized in 6 patients (27%). All six individuals harbored the D30N mutation, and none presented the L90M mutation. Other PI resistance mutations were present in 5 subjects (at codons 36, 63, 71, 77, 82 and/or 88). Secondary PI resistance mutations were present in another 9 subjects. By contrast, mutations conferring resistance to reverse transcriptase inhibitors were present in 50% of the patients, and the M184V substitution was the most frequently seen. CONCLUSIONS: Nearly 75% of patients failing their first PI-based triple regimen containing NFV do not harbor PI resistance mutations. The D30N substitution, rather than L90M, is the most frequently recognized, which does not challenge the efficacy of further rescue interventions with other PIs. This observation supports the use of nelfinavir as first protease inhibitor.