Cell-specific alterations in Pitx1 regulatory landscape activation caused by the loss of a single enhancer

Developmental genes are frequently controlled by multiple enhancers sharing similar specificities. As a result, deletions of such regulatory elements have often failed to reveal their full function. Here, we use the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion of one of its enhancers (Pen). By combining single cell transcriptomics and an in-embryo cell tracing approach, we observe an increased fraction of Pitx1 non/low-expressing cells and a decreased fraction of Pitx1 high-expressing cells. We find that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. This locus mis-activation induces a localized heterochrony and a concurrent loss of irregular connective tissue, eventually leading to a clubfoot phenotype. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes

Context-dependent enhancer function revealed by targeted inter-TAD relocation

The expression of some genes depends on large, adjacent regions of the genome that contain multiple enhancers. These regulatory landscapes frequently align with Topologically Associating Domains (TADs), where they integrate the function of multiple similar enhancers to produce a global, TAD-specific regulation. We asked if an individual enhancer could overcome the influence of one of these landscapes, to drive gene transcription. To test this, we transferred an enhancer from its native location, into a nearby TAD with a related yet different functional specificity. We used the biphasic regulation of Hoxd genes during limb development as a paradigm. These genes are first activated in proximal limb cells by enhancers located in one TAD, which is then silenced when the neighboring TAD activates its enhancers in distal limb cells. We transferred a distal limb enhancer into the proximal limb TAD and found that its new context suppresses its normal distal specificity, even though it is bound by HOX13 transcription factors, which are responsible for the distal activity. This activity can be rescued only when a large portion of the surrounding environment is removed. These results indicate that, at least in some cases, the functioning of enhancer elements is subordinated to the host chromatin context, which can exert a dominant control over its activity.</p

Pre-hypertrophic chondrogenic enhancer landscape of limb and axial skeleton development

Chondrocyte differentiation controls skeleton development and stature. Here we provide a comprehensive map of chondrocyte-specific enhancers and show that they provide a mechanistic framework through which non-coding genetic variants can influence skeletal development and human stature. Working with fetal chondrocytes isolated from mice bearing a Col2a1 fluorescent regulatory sensor, we identify 780 genes and 2'704 putative enhancers specifically active in chondrocytes using a combination of RNA-seq, ATAC-seq and H3K27ac ChIP-seq. Most of these enhancers (74%) show pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. Notably, genetic variations overlapping these enhancers better explain height differences than those overlapping non-chondrogenic enhancers. Finally, targeted deletions of identified enhancers at the Fgfr3, Col2a1, Hhip and, Nkx3-2 loci confirm their role in regulating cognate genes. This enhancer map provides a framework for understanding how genes and non-coding variations influence bone development and diseases

Nutrition management and early rehabilitation in ICU pregnant with hyperemesis gravidarum complicated by central pontine myelinolysis: A case report

Summary: Our case report aims to highlight the multidisciplinary approach adopted for the avoidance of ICU-acquired weakness and the assessment of nutritional therapy in 16 weeks young pregnant with diagnosis of hyperemesis gravidarum complicated with central pontine myelinolysis, after the development of acute respiratory failure due to pneumonia.Thiamine and electrolytes were properly supplemented to minimize the high risk of developing refeeding syndrome. Due to severe nausea and vomiting, antiemetic therapy was started and a parenteral route was chosen during the first two days of non-invasive ventilation. On day three, the patient was intubated and mechanically ventilated due to severe respiratory failure, semi-elemental formula was started by enteral route while parenteral nutrition was supplemented and early rehabilitation was started. Antiemetic therapy was continued until day 28th when the pregnant woman was shifted to oral nutrition only. On day 87th the postpartum mother was transferred to the rehabilitation center for an intensive rehabilitative program based on the motor recovery of lower limb and swallowing recovery

Effects of Recruitment Maneuver and Positive End-expiratory Pressure on Respiratory Mechanics and Transpulmonary Pressure during Laparoscopic Surgery

Background: The authors tested the hypothesis that during laparoscopic surgery, Trendelenburg position and pneumoperitoneum may worsen chest wall elastance, concomitantly decreasing transpulmonary pressure, and that a protective ventilator strategy applied after pneumoperitoneum induction, by increasing transpulmonary pressure, would result in alveolar recruitment and improvement in respiratory mechanics and gas exchange. Methods: In 29 consecutive patients, a recruiting maneuver followed by positive end-expiratory pressure 5 cm H2O maintained until the end of surgery was applied after pneumoperitoneum induction. Respiratory mechanics, gas exchange, blood pressure, and cardiac index were measured before (TBSL) and after pneumoperitoneum with zero positive end-expiratory pressure (TpreOLS), after recruitment with positive end-expiratory pressure (TpostOLS), and after peritoneum desufflation with positive end-expiratory pressure (Tend). Results: Esophageal pressure was used for partitioning respiratory mechanics between lung and chest wall (data are mean ± SD): on TpreOLS, chest wall elastance and elastance of the lung (EL) increased (8.2 ± 0.9 vs. 6.2 ± 1.2 cm H2O/L, respectively, on TBSL; P = 0.00016; and 11.69 ± 1.68 vs. 9.61 ± 1.52 cm H2O/L on TBSL; P = 0.0007). On TpostOLS, both chest wall elastance and EL decreased (5.2 ± 1.2 and 8.62 ± 1.03 cm H2O/L, respectively; P = 0.00015 vs. TpreOLS), and PaO2/inspiratory oxygen fraction improved (491 ± 107 vs. 425 ± 97 on TpreOLS; P = 0.008) remaining stable thereafter. Recruited volume (the difference in lung volume for the same static airway pressure) was 194 ± 80 ml. PplatRS remained stable while inspiratory transpulmonary pressure increased (11.65 + 1.37 cm H2O vs. 9.21 + 2.03 on TpreOLS; P = 0.007). All respiratory mechanics parameters remained stable after abdominal desufflation. Hemodynamic parameters remained stable throughout the study. Conclusions: In patients submitted to laparoscopic surgery in Trendelenburg position, an open lung strategy applied after pneumoperitoneum induction increased transpulmonary pressure and led to alveolar recruitment and improvement of chest wall elastance, EL, and gas exchange

Peep titration based on the open lung approach during one lung ventilation in thoracic surgery: a physiological study

Abstract Background During thoracic surgery in lateral decubitus, one lung ventilation (OLV) may impair respiratory mechanics and gas exchange. We tested a strategy based on an open lung approach (OLA) consisting in lung recruitment immediately followed by a decremental positive-end expiratory pressure (PEEP) titration to the best respiratory system compliance (CRS) and separately quantified the elastic properties of the lung and the chest wall. Our hypothesis was that this approach would improve gas exchange. Further, we were interested in documenting the impact of the OLA on partitioned respiratory system mechanics. Methods In thirteen patients undergoing upper left lobectomy we studied lung and chest wall mechanics, transpulmonary pressure (PL), respiratory system and transpulmonary driving pressure (ΔPRS and ΔPL), gas exchange and hemodynamics at two time-points (a) during OLV at zero end-expiratory pressure (OLVpre-OLA) and (b) after the application of the open-lung strategy (OLVpost-OLA). Results The external PEEP selected through the OLA was 6 ± 0.8 cmH2O. As compared to OLVpre-OLA, the PaO2/FiO2 ratio went from 205 ± 73 to 313 ± 86 (p = .05) and CL increased from 56 ± 18 ml/cmH2O to 71 ± 12 ml/cmH2O (p = .0013), without changes in CCW. Both ΔPRS and ΔPL decreased from 9.2 ± 0.4 cmH2O to 6.8 ± 0.6 cmH2O and from 8.1 ± 0.5 cmH2O to 5.7 ± 0.5 cmH2O, (p = .001 and p = .015 vs OLVpre-OLA), respectively. Hemodynamic parameters remained stable throughout the study period. Conclusions In our patients, the OLA strategy performed during OLV improved oxygenation and increased CL and had no clinically significant hemodynamic effects. Although our study was not specifically designed to study ΔPRS and ΔPL, we observed a parallel reduction of both after the OLA. Trial registration TRN: ClinicalTrials.gov , NCT03435523 , retrospectively registered, Feb 14 2018

Physiological effects of the open lung approach during laparoscopic cholecystectomy:focus on driving pressure

BACKGROUND: During laparoscopy, respiratory mechanics and gas exchange are impaired because of pneumoperitoneum and atelectasis formation. We applied an open lung approach (OLA ) consisting in lung recruitment followed by a decremental positive-end expiratory pressure (PEE P) trial to identify the level of PEE P corresponding to the highest compliance of the respiratory system (best PEE P). Our hypothesis was that this approach would improve both lung mechanics and oxygenation without hemodynamic impairment. METHODS: We studied twenty patients undergoing laparoscopic cholecystectomy. We continuously recorded respiratory mechanics parameters throughout a decremental PEE P trial in order to identify the best PEE P level. Furthermore, lung and chest wall mechanics, respiratory and transpulmonary driving pressures (ΔP), gas exchange and hemodynamics were recorded at three time-points: 1) after pneumoperitoneum induction (TpreOLA ); 2) after the application of the OLA (TpostOLA ); 3) at the end of surgery, after abdominal deflation (Tend). RESULTS : The “best PEE P” level was 8.1±1.3 cmH2O (range 6 to 10 cmH2O), corresponding to the highest compliance of the respiratory system (CRS ). This “best PEEP” level corresponded with lowest ΔPL. OLA increased the compliance of the lung and of the chest wall, and decreased ΔPRS and ΔPL. PaO2/FiO2 increased from 299±125 mmHg to 406±101 mmHg (P=0.04). Changes in respiratory mechanics, driving pressures and oxygenation were maintained until Tend. Hemodynamic parameters remained stable throughout the study period. CONCLUSIONS: In patients undergoing laparoscopic cholecystectomy, the OLA was suitable for bedside PEE P setting, improved lung mechanics and gas exchange without significant adverse hemodynamic effects

Effects of Ciprofloxacin Alone or in Mixture with Sulfamethoxazole on the Efficiency of Anaerobic Digestion and Its Microbial Community

Some livestock farms rely on anaerobic digestion (AD) technology for manure disposal, thus obtaining energy (biogas) and fertilizer (digestate). Mixtures of antibiotics used for animal health often occur in organic waste and their possible synergistic/antagonistic effects on microorganisms involved in AD are still poorly studied. This work focuses on the effects of adding ciprofloxacin, alone (5 mg L−1) and in combination with sulfamethoxazole (2.5–5–10 mg L−1), on AD efficiency and microbial community structure. The experiment consisted of 90-day cattle manure batch tests and antibiotic removal percentages were assessed. Adding antibiotics always promoted CH4 and H2 production compared to untreated controls; however, CH4 production was lowered with the highest ciprofloxacin (CIP) concentrations. The overall results show antibiotic degradation caused by acidogenic Bacteria, and CH4 was mainly produced through the hydrogenotrophic-pathway by methanogenic Archaea. Shifts in microbial community abundance (DAPI counts) and composition (Illumina-MiSeq and FISH analyses) were observed

Long-term benefits of dapagliflozin on renal outcomes of type 2 diabetes under routine care: a comparative effectiveness study on propensity score matched cohorts at low renal risk

Background: Despite the overall improvement in care, people with type 2 diabetes (T2D) experience an excess risk of end-stage kidney disease. We evaluated the long-term effectiveness of dapagliflozin on kidney function and albuminuria in patients with T2D. Methods: We included patients with T2D who initiated dapagliflozin or comparators from 2015 to 2020. Propensity score matching (PSM) was performed to balance the two groups. The primary endpoint was the change in estimated glomerular filtration rate (eGFR) from baseline to the end of observation. Secondary endpoints included changes in albuminuria and loss of kidney function. Findings: We analysed two matched groups of 6197 patients each. The comparator group included DPP-4 inhibitors (40%), GLP-1RA (22.3%), sulphonylureas (16.1%), pioglitazone (8%), metformin (5.8%), or acarbose (4%). Only 6.4% had baseline eGFR &lt;60&nbsp;ml/min/1.73&nbsp;m2 and 15% had UACR &gt;30&nbsp;mg/g. During a mean follow-up of 2.5 year, eGFR declined significantly less in the dapagliflozin vs comparator group by 1.81&nbsp;ml/min/1.73&nbsp;m2 (95% C.I. from 1.13 to 2.48; p&nbsp;&lt;&nbsp;0.0001). The mean eGFR slope was significantly less negative in the dapagliflozin group by 0.67&nbsp;ml/min/1.73&nbsp;m2/year (95% C.I. from 0.47 to 0.88; p&nbsp;&lt;&nbsp;0.0001). Albuminuria declined significantly in new-users of dapagliflozin within 6 months and remained on average 44.3&nbsp;mg/g lower (95% C.I. from&nbsp;-66.9 to&nbsp;-21.7; p&nbsp;&lt;&nbsp;0.0001) than in new-users of comparators. New-users of dapagliflozin had significantly lower rates of new-onset CKD, loss of kidney function, and a composite renal outcome. Results were confirmed for all SGLT2 inhibitors, in patients without baseline CKD, and when GLP-1RA were excluded from comparators. Interpretation: Initiating dapagliflozin improved kidney function outcomes and albuminuria in patients with T2D and a low renal risk. Funding: Funded by the Italian Diabetes Society and partly supported by a grant from AstraZeneca