K. pneumoniae (CP-Kp) has been established as important nosocomial pathogen in most tertiary care hospitals in Greece. The aim of the present study was to examine the impact of an enhanced infection control program on the containment of CP-Kp in a haematology unit where the incidence of CP-Kp infections was high.Carbapenemase-producing The study was conducted from June 2011 to December 2014 in a haematology unit of a tertiary-care 500-bed hospital located in Athens, Greece. A bundled intervention  was tested whether would reduce colonization and infection caused by CP-Kp.p < 0.001 for linear trend), the hospitalization prevalence (p = 0.001 for linear trend) and the incidence rate of CP-Kp colonization (p = 0.072 for linear trend) were declining. Application of segmented linear regression revealed that both the change in the level of CP-Kp BSI incidence rates (p = 0.001) as well as the difference between pre- and post-intervention slopes were statistically significant (p < 0.001).A total of 2507 rectal swabs were obtained; 1199 upon admission from June 2011 to June 2013 and 1307 during hospitalization from June 2011 to December 2012. During intervention the admission prevalence of CP-Kp colonization (A bundled intervention including active surveillance cultures on admission can attain maximum containment of CP-Kp colonization and infection in endemic acute healthcare settings. K. pneumoniae (CP-Kp) has been established as important nosocomial pathogen worldwide [R0, is estimated to be 2, a number sufficient to trigger outbreaks in the healthcare setting [Carbapenemase-producing orldwide , 2. A hiorldwide , 4. In h setting . In addiInfection and colonization rates by CP-Kp strains in Greek tertiary care hospitals are considered amongst the highest in Europe . The dirKlebsiella spp, Acinetobacter spp and Pseudomonas spp. However, the escalating shortages in human and financial resources in many of our hospitals do not allow the full and proper implementation of the required measures in a sustainable and timely fashion [Recognizing the urgent need to reduce the dispersion of CP-pathogens, the Hellenic Center for Disease Control and Prevention has drawn up an action plan for the containment of infections caused by multi-drug resistant organisms in health-care facilities . The bas fashion .The application of mathematical modeling in the data of a previous observational study conducted in a unit of our hospital  where CP-Kp endemicity was sustained by influx of carriers and cross-transmission, indicated the pivotal role of active surveillance cultures in controlling CP-Kps . Based oThe study was conducted from June 2011 to December 2014 in a 31-bed haematology unit of a tertiary-care 500-bed hospital located in Athens, Greece. The unit is consisted of fourteen rooms; seven 3-bed, three 2-bed and four single rooms. The vast majority of patients admitted in the unit are patients with haematologic malignancies. The mean nurse to patient ratio was 1:10 during the morning shift and 1:15 during afternoon and night shifts. Compliance of healthcare workers (HCWs) with hand hygiene before intervention was ranging from 10 to 20%. After January 2011 an increasing number of CP-Kp BSIs was observed in the unit. A point prevalence for rectal colonization was conducted among 28 patients hospitalized in the unit which revealed that 8 (28.6%) patients were colonized with CP-Kp. These observations prompted the infection control committee to design and implement an enhanced infection control program.Before implementing the bundled intervention in June 2011, all healthcare personnel attended an educational course. Training and feedback were provided from the infection control team once a month initially and every 3 months thereafter. Hand-washing basins and dispensers containing alcohol-based disinfectants were located near all beds. Personal protective items including gloves and gowns and dedicated patient-care equipment were made available in the unit. Also, written instructions were given to all patient relatives. The infection control measures included i) hand hygiene, ii) active surveillance cultures of rectal swabs within 24 h after admission and weekly thereafter for all patients staying for more than 24 h, iii) separation of carriers or infected patients in single rooms or cohorting in two- or three-bed rooms, iv) staff cohorting to care for patients known to harbour CP-Kp, when feasible, v) contact precautions, and vi) environmental cleaning of patient-room twice daily. The weekly follow-up surveillance cultures during hospitalization were performed until the CP-Kp transmission was considered negligible (December 2012) and the surveillance cultures on admission were continued up to June 2013  or not performed, according to the “My five moments for hand hygiene” WHO Hand Hygiene Improvement Strategy. A mean of 200 observations were performed every month. Compliance to isolation (single room or cohorting) of colonized/infected patients, the assignment of dedicated nursing staff for carriers, as well as the implementation of contact precautions were monitored daily.K. pneumoniae were examined for production of carbapenemases by combined disk synergy test [blaVIM, blaKPC, blaNDM, and blaOXA-48) were detected by polymerase chain reaction and the genetic relatedness of the isolates was examined by pulsed-field gel electrophoresis (PFGE), as described previously [Rectal swabs were obtained and inoculated on McConkey agar No.3 plates (OXOID) containing 0.5 mg/L of meropenem and incubated at 37 °C for a maximum of 48 h. Isolates identified as rgy test . Carbapeeviously .As primary outcome of the intervention was the incidence of CP-Kp BSIs. Secondary outcomes included the incidence and prevalence of CP-Kp colonization of hospitalized patients, the admission prevalence of CP-Kp colonization and the incidence of BSIs due to any pathogen. A patient was defined as colonized if he/she was found positive for CP-Kp in a surveillance culture. A patient found positive in the first surveillance culture upon admission was considered to be already colonized before entering the unit, whereas when a patient found CP-Kp negative upon admission and positive in any of the follow-up rectal swab cultures was considered as being colonized during hospitalization. A colonized patient was thought to be decolonized if the investigators had recorded at least three consecutive rectal samples to be CP-Kp negative. CP-Kp BSI was defined as detection of CP-Kp in a blood culture along with consistent clinical findings. Onset of BSI was defined as the date of collection of the first blood culture that yielded CP-Kp.CP-Kp colonization on admission was assed based on rectal swabs obtained within 24 h since patient’s admission. The admission prevalence of CP-Kp colonization per month was then estimated as the proportion of positive rectal swabs out of the total number of swabs collected upon admission during that month. We also estimated the hospitalization prevalence of CP-Kp colonisation per month as the proportion of colonised patients out of the total number of hospitalised patients in the unit during that month (patients with at least one positive swab were considered colonized). We assessed the post-intervention slope of the linear trend in the monthly CP-Kp colonisation prevalence on admission and during hospitalisation using linear regression. We estimated the incidence rate of CP-Kp colonization during hospitalization per month. Only patients who were not colonized on admission and had at least two available swabs per admission, one upon admission and at least one during hospitalization, were included in the analysis. The person-time was calculated as the time between admission and discharge for those who remained un-colonised, or as the time between admission and the midpoint between the last negative and first positive swab for those who were colonized during hospitalization. The incidence rate of colonization was then estimated as the number of new colonisations occurring during hospitalization divided by the total person-time at risk for that one-month period. We further estimated the incidence of BSIs (bimonthly or quarterly) as the number of new infections occurring in the specific period over the total patient-time at risk in that period. Similarly, we assessed the incidence of bloodstream infections caused by pathogens other than CP-Kp. As in the case of CP-Kp BSI incidence, there were data available during a long pre-intervention period. Thus, it was possible to assess the impact of the intervention using the longitudinal data series on BSI incidence before and after its implementation in June 2011 using segmented linear regression. This method allowed to estimate the change in level of BSI incidence rates immediately after the intervention and the difference between pre- and post-intervention slopes of the outcome trend.A total of 2005 patients were admitted in the unit; 1199 from June 2011 to June 2013 and 806 from July 2013 to December 2014. The mean hospital stay was 15 days and the median 9 days. Overall, 2507 rectal swabs were obtained; 1199 upon admission from June 2011 to June 2013 and 1307 during hospitalization from June 2011 to December 2012. The average post-intervention monthly compliance with hand hygiene among the medical and nursing staff was 60 and 50%, respectively . High level of adherence was observed in isolation/cohorting (100% of CP-Kp colonized/infected patients) and in contact precautions (100%). Given the low nurse to patient ratio, dedicated nursing staff was assigned only during the morning shift.p < 0.001) and the number of new CP-Kp carriers decreasing . The colonization incidence during hospitalization was 10.6/1000 patient-days in June 2011 and declined to 0/1000 patient-days in December 2012 , indicating negligible cross transmission . Moreover, a decline in the incidence of BSIs caused by pathogens other than CP-Kp was observed  , 7 Verona integron-encoded metallo-beta-lactamase (VIM), one New Delhi metallo-beta-lactamase (NDM-1) and one isolate both type of enzymes, VIM and KPC. Regarding the BSI isolates, nine produced KPC, one VIM and one NDM. A total of 67 K. pneumoniae isolates were studied by PFGE, 57 obtained from surveillance cultures (more than one isolates per patient were available from 18 of 38 carriers) and 10 from blood cultures. Fifty-four organisms produced KPC, nine VIM, three NDM and one isolate, both type of enzymes, VIM and KPC. PFGE typing revealed 15 different genomic profiles. Forty-six of 54 KPC isolates were grouped in one PFGE type, while the remaining 8 isolates displayed six distinct profiles containing 1 to 2 isolates each. The nine VIM-producers exhibited seven different PFGE types, each one containing 1 to 2 isolates and the three NDM-producers consisted a distinct group. The isolate that carried both blaVIM and blaKPC genes was classified in one of the KPC PFGE types. Of note, all BSIs were caused by CP-Kp exhibiting identical PFGE type with that of the colonizing organism  produced mase NDM- and one The findings presented herein demonstrate that early identification of asymptomatic CP-Kp carriers followed by physical separation from non-carriers in conjunction with contact precautions, improved compliance in hand hygiene, and environmental cleaning, had a significant effect on containing CP-Kp spread and on reducing CP-Kp BSIs in an haematology unit where the incidence of CP-Kp infections and the associated mortality were high.Prior to intervention a substantial proportion (28.6%) of hospitalized patients in the unit were colonized with CP-Kp. Apparently, this was result of i) constant inflow of CP-Kp colonized patients and ii) transmission from patient to patient due to inadequate infection control practices. Indeed, approximately 10% of the admitted patients were already colonized and a large proportion of CP-Kp isolates examined by PFGE was classified in groups with identical genetic profiles, indicating ongoing horizontal transmission. In health care facilities with such characteristics, the infection control strategy should aim to interrupt both chains of transmission: i) the inflow of carriers by early identification and physical separation and ii) the horizontal transmission by contact precautions and enhanced standard infection control practices.Transmission from patient to patient via the hands of health care workers is the main route of spread in the acute-care setting . In a moUnrecognized CP-Kp carriers can serve as a potential source of transmission to other patients. Previous studies have shown that the use of clinical cultures may fail to detect a significant proportion of colonized patients and only after employing active surveillance the incidence of CP-Kp infections declined , 17. SimContainment of carbapenemase-producing Enterobactericeae (CRE) has been demonstrated by several studies after applying infection control interventions in the form of bundles , 18–25. Previous studies have shown that a substantial proportion of CP-Kp colonized haematologic patients may develop bacteremia with detrimental effects for the host –32. In oHerein was shown that bundled intervention, including active surveillance, can attain maximum containment of CP-Kp colonization and infection in endemic acute healthcare settings. Considering the impact of CP-Kp infections on human lives, a multifaceted intervention programme to prevent and control such infections should be in place in all acute health care facilities. When a hospital-wide intervention is not feasible due to limited resources, there is need for prioritization and prompt implementation of a rigorous and effective program in “high risk” units to protect the most vulnerable patients.


As per the literature, patients with intraventricular conduction delay (IVCD) do not respond well to cardiac resynchronization therapy (CRT) alone. They need advanced technological approach and out of the box thinking for a good response.Ours is a case of ischemic cardiomyopathy with wide QRS-IVCD, a non-responder to CRT. While planning for replacement of the device for early replacement indicator (ERI), we decided to do His-optimized CRT/left bundle optimized CRT (HOT-CRT/LOT-CRT) for the patient.The challenges we faced with the present available hardware paved a way for insisting on the limitation of the available lumenless lead to penetrate calcified the septum and importance of the pre-procedure evaluation of intraventricular septum (IVS) for calcification by more than just echocardiography. Cardiac resynchronization therapy (CRT) has widely been used in patients of symptomatic chronic heart failure (HF) with wide QRS complex, refractory to optimal medical therapy and having left ventricular ejection fractionLVEF is < 35%. However, one third of the patients are non-responders and do not respond to this therapy. Physiological pacing, i.e., permanent His bundle pacing (HBP)/left bundle branch pacing (LBBP) is proving a promising alternative to biventricular pacing (BiVP) for some of these challenging patients. In this report, we present a case of HOT-CRTD with good outcome of physiological pacing despite challenges.A 64-year-old male patient with a known history of coronary artery disease had undergone a CRTD implant in November 2015 for IVCD with wide QRS (159 ms), QLV was 102 ms  junction was seen Fig.  a. A fluThe HB lead was attached in RV pacing port (DF1 RV lead), coronary sinus (CS) lead in LV port, and RA lead in atrial port in the new pulse generator. Previous RV pace-sense lead terminal was buried deep in the pocket. The pre-pectoral device pocket was closed in three layers; DFT (defibrillation threshold test) was not done as per protocol.The strategy of fused complex was employed whereby LV pacing timing was delayed, relative to HBP output Fig. . A narroAbout 30–50% of patients with HF who meet the criteria do not benefit by CRT . In addiThrough this case, we demonstrated that HOT-CRT is effective in correcting IVCD. It is an option particularly in patients who are CRT non-responders. Success of LBBP is dependent on IVS anatomy. Local dense scar or calcification at the septum may result in failure of LBBP. The present 3830 SelectSecureTM active pacing lead  used for LBBP has limited penetration capability. Focused hardware needs to be developed to overcome such impediment.


The current global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has magnified the risk to healthcare providers when inititiating airway management, and safe tracheal intubation has become of paramount importance. Mitigation of risk to frontline providers requires airway management to be an orchestrated exercise based on training and purposeful simulation. Role allocation and closed-loop communication form the foundation of this exercise. We describe a methodical, 10-step approach from decision-making and meticulous drug and equipment choices to donning of personal protective equipment, and procedural concerns. This bundled approach will help reduce unplanned actions, which in turn may reduce the risk of aerosol transmission during airway management in resource-limited settings. The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or COVID-19, pandemic has brought infections that are transmitted via droplet and aerosol under the spotlight.Rapid sequence intubation (RSI) and invasive mechanical ventilation are preferred. Non-invasive positive pressure ventilation (NIPPV) increases the risk of aerosol generation; NIPPV has been associated wth increased risk of healthcare worker infection and hence should be avoided.6The care area is divided as follows:Hot zone: A three-meter [9.85 feet] radius around the patientWarm zone: The area between hot and cold zone where decontamination takes placeCold zone: The outermost noncontaminated area.The intubation team members are described in Encourage closed-loop communication.What do we already know about this issue?Several protocols for intubation during the COVID-19 pandemic aim at reducing transmissibility of infections by using sophisticated equipment.What was the research question?How can we reduce the risk of aerosol hazards from infectious diseases transmitted during intubation?What was the major finding of the study?Execution of intubation in suspected aerosol-transmitted infections can be performed systematically in low-resource settings.How does this improve population health?The protocol is aimed at safeguarding healthcare professionals against aerosol hazards while performing airway interventions.Pre-assessment phase and pre-briefing phase – Cold ZoneStep a. Review patient clinical data to determine appropriateness of endotracheal intubation and mechanical ventilation for the patient.Step b. Team leader (TL) debriefs the intubation plan to the team to avoid unplanned and unarticulated actions.Step c. Infection control nurse (ICN) alerts team to any breach of protocol or infection control practice.Preparatory phase – Cold ZoneStep a. Use continous positive airway pressure mode with non-invasive ventilation (NIV) mask for preoxygenation. The registered respiratory therapist (RRT) assembles the ventilator with its circuit including preparation of the NIV mask with a viral filter and checks for possible leaks and disconnections. Ventilator settings: pressure support 0 centimeters of water, positive end expiratory pressure as per the requirement, and fraction of inspired oxygen to 100%. Deselect the apnea setting.Step b. Review the equipment required for intubation  Table 2Table 2; Step c. The assembly  of the ePreoxygenation Phase: Hot ZoneStep a: The RRT ensures wall-mounted suction unit is properly connected. A Yankauer suction connected to the wall-mounted suction unit should be available, but its usage should be judicious. The suction tip, if used, should be disposed of in a Ziploc bag.Step b: TL at the head of the bed places the NIV mask with the viral filter onto the patient and ensures proper sealing to avoid leak. The RRT “starts” the ventilator and preoxygenates until adequate oxygen saturation is attained. Avoid bag-valve mask for preoxygenation. Meticulous preoxygenation should be done for 3–5 minutes.Step c: RN ensures patent intravenous access, assesses the vitals periodically, and communicates them to the TL.Peri Induction phase: Hot ZoneStep a: RN administers the pre-calculated dose of the induction agent followed by the paralytic agent to the patient.Step b: Approrpiate patient positioning should be performed to maximise safe apnoea time.Peri-intubation Phase: Hot ZoneStep a: The RRT sets the ventilator on standby mode b after adequate paralysis and oxygen saturation is achieved.Step b: TL subsequently removes the NIV mask, which is disconnected from the ventilator by the RRT and placed in a Ziploc bag.Step c: TL performs laryngoscopy. During this time, the RRT is required to change the settings of the ventilator to “assist control mode ventilation.”. Once the vocal cords are visualized, the RRT hands over the intubating unit to the TL who should then pass the bougie between the cords under direct visualization. Video laryngoscope is a preferred choice for intubation of such patients, if available.Step d: The RRT assists in guiding the ET over the bougie and should subsequently inflate the cuff with the pre-filled air syringe.Step e: The RRT then removes the bougie and disposes of it in the preset disposition system .Step f: The RRT then proceeds to connect the ET to the ventilator and convert the ventilator from standby to its preset settings. Simultaneously, the TL removes the laryngoscope and places it in a Ziploc bag.Step g: The RN confirms the position of the tube with five-point auscultation, following which the stethoscope should be disposed of in the Ziploc bag. End-tidal carbon dioxide confirmation is advised, if available.Post-intubation Phase: Hot ZoneStep a: Continue ventilation and monitor hemodynamics. Initiate early sedation and analgesia.Step b: Ensure all soiled equipment has been disposed of appropriately into the yellow bag for decontamination .88Step c: The order of doffing and decontamination is TL, followed by the RN, and then the RRT who should be separately overviewed and monitored by the ICN.This sequence should guide healthcare professionals to minimize aerosol and droplet transmission during intubation and expedite better patient care. This approach does not involve significant resource intensification and can be done in resource-limited settings.


P < 0.05) in the retinas of OIR mice compared with controls. Validation by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in four selected tsRNAs was consistent with the results of small RNA sequencing. Bioinformatics analyses identified 153 altered target genes of the four validated tsRNAs. These altered target genes were largely enriched in developmental process, cell periphery and protein binding, as well as Th1 and Th2 cell differentiation pathway. Our study suggests tsRNAs play key roles in the pathogenesis of RNV, indicating their therapeutic potential to treat patients with RNV. Moreover, small RNA sequencing is a useful tool to identify changes in tsRNA expression, an important indicator of the progress of retinal diseases.Retinal neovascularization (RNV) is characterized in retinopathy of prematurity (ROP), diabetic retinopathy (DR), and retinal vein occlusion (RVO), which leads to severe vision loss and even blindness. To reveal the altered transfer RNA-derived small RNA (tsRNA)s in RNV, and to investigate the underlying mechanisms of the altered tsRNAs involved in RNV, we carried out a small RNA sequencing to profile tsRNA expressions in the retinas of mice with oxygen-induced retinopathy (OIR) and control mice. A total of 45 tsRNAs were significantly changed (fold change ≥ 1.5 and  Retinal neovascularization (RNV) is characterized in several kinds of retinal diseases, such as retinopathy of prematurity (ROP), diabetic retinopathy (DR), and retinal vein occlusion (RVO), which leads to severe vision loss and even blindness Although anti-vascular endothelial growth factor (VEGF) treatment has shown great benefits to patients with RNV Li et al. discussed the role of tsRNAs plays in ischemic pathophysiology and in post-ischemia angiogenesis. The study showed that angiogenesis could be suppressed by the tRNAVal(CAC)- and tRNAGly(GCC)-derived small RNAs and up-regulated these fragments in ischemic tissues may alleviate angiogenesis Transfer RNAs (tRNAs) were recently found to be an important source of a group of functional non-coding RNAs, tRNA-derived small RNAs, including tRNA-derived RNA fragments (tRFs) and tRNA-derived stress-induced RNAs (tiRNAs), both of which are differentiated from each other according to the cleavage position of the mature or precursor tRNAs C57BL/6J mice  were used in the study. Mice were treated by the rules of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures were subjected to approval by the Institutional Animal Care and Use Committee of Central South University, China .The mouse model of OIR was established as previously described TM instrument  were used to assess the quantity and integrity of RNA samples.Retinas from both eyes of each mouse were mixed as a sample. RNA was isolated by Trizol RNA extraction kit  followed by instructions of the manufacturer. Agarose gel electrophoresis as well as NanodropPretreatment of tsRNA and library preparation, small RNA sequencing, collection and analysis of the data were conducted as described Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to confirm the selected tsRNAs' sequencing results. RNA was treated by rtStar™ tRF&tiRNA Pretreatment Kit  and then converted to cDNA using rtStar™ First-Strand cDNA Synthesis Kit (3' and 5' adaptor) (Arraystar). qRT-PCR experiments were performed on ViiA 7 Real-time PCR System  by using 2 × PCR Master Mix (Arraystar). Reaction conditions were described in our previous study P < 0.05) were selected to construct the network according to the data of our previous microarray analysis study (25). Gene Ontology (GO) (http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) were used for prediction of biological functions and pathways involved in RNV.Prediction of target genes of four tsRNAs were conducted by miRanda and Targetscan. The significantly altered mRNAs with a threshold  was applied for screening significantly altered tsRNAs. The statistical difference of qRT-PCR was assessed by Student's t-test (P < 0.05) and showed as the mean ± SEM.The small RNA sequencing data of tsRNAs was evaluated by counts per million (CPM) mapped reads. R package edgeR Followed by small RNA sequencing, raw sequencing data of tsRNAs were analyzed based on alignment statistical analysis. A 3D visualization of the relationship between the samples was given by a principal component analysis (PCA) plot, which indicated a distinguishable tsRNA expression profiling among these included samples, and also suggested a clear difference between the two groups in the molecular composition Figure a.To evaluate the difference of expression level of tsRNAs in the retina of OIR and controls, a threshold of the fold change of ≥1.5 and p-value < 0.05 was applied, which resulted in significantly changed tsRNAs in a hierarchical clustering heat map Figure .A Venn diagram was plotted with R VennDiagram package to investigate the shared and specifically expressed tsRNAs between the retina of OIR and controls. The results showed that there were 444 tsRNAs shared between both groups, while 74 and 42 tsRNAs were uniquely expressed in OIR and controls, respectively  were randomly selected. qRT-PCR results showed a consistent trend with small RNA sequencing results Figure a. The exAccording to the tsRNA-target gene network based on TargetScan and miRanda, we identified that there were 2252 target genes associated with four validated tsRNAs. These target genes were intersected with significantly changed mRNAs with a threshold of ≥1.5-fold change and a p-value of <0.05 in OIR mice according to a microarray analysis in our previous study Analyses of GO and KEGG pathway analyses were performed according to the predicted target genes with a threshold of ≥1.5-fold change and a p-value of <0.05 to further explore the possible involved functions and pathways of these altered tsRNAs. The most enriched oncology in three included domains  are developmental process GO: 00032502), cell periphery (GO: 0071944) and protein binding (GO: 0005515) Figure a and b. 2502, celAlthough increasing evidence shows that miRNAs and tsRNAs play crucial roles in choroidal neovascularization Similar to miRNAs, tsRNAs are able to regulate gene expressions at post-transcriptional level through binding corresponding sequencing in mRNAs Our small RNA sequencing data suggested that there were significant differences in retinal tsRNA expression between OIR mice and controls. GO analysis implied that target genes of altered tsRNAs involved in developmental process, indicating that tsRNAs may also play some roles in developmental process of RNV. tsRNAs might also be involved in protein binding, implying tsRNA could be a mediator to regulate protein interaction and contribute to RNV formation, which is similar to the mechanisms of cancer studies Network analysis identified that Tspan15 is the only up-regulated gene targeted by tRF-Phe-GAA-001 and tRF-Lys-TTT-029, both of which were up-regulated in OIR mice. A study in hepatocellular carcinoma reported that cell proliferation is significantly increased by Tspan15 overexpression in HepG2 cell line led to cell proliferation by regulating abundant membrane proteins to promote expression of growth factor receptors The KEGG pathway analysis demonstrated that Th1 and Th2 cell differentiation pathway is the most enriched and reliable pathway involving altered target genes of four validated altered tsRNAs. As we reported in an earlier study, M2 macrophages increased a larger extent than M1 macrophages in the retinas of OIR mice As we discussed before, tRFs can be induced specifically by hypoxia, making it a novel type of regulatory factors as well as therapeutic targets In this study, we found a total of 45 tsRNAs were significantly altered in OIR mice compared to controls, which demonstrated a dysregulation of tsRNA in OIR mouse model and RNV related diseases. In addition, through bioinformatics analyses, we identified numerous targeted genes that are related with four validated altered tsRNAs. Furthermore, by GO and KEGG analyses, we detected several potential biological functions and possible involved pathways that might contribute to RNV pathogenesis, which also provided potential novel targets for therapies of retinal neovascular diseases.There are several limitations in the study. First of all, the expression profiles of the tsRNAs in variable period of OIR pathogenesis have not been examined. It has been shown that neovascular regression occurs from P17 to P25 in OIR mouse model In summary, we identified altered tsRNAs in the retinas of OIR mice and predicted potential target genes which might be involved in RNV pathogenesis. All these preliminary data revealed that tsRNAs could affect and participate in the pathological processes in OIR mouse model and RNV formation, suggesting that tsRNAs could serve as potential biomarkers to identify retinal neovascular diseases, as well as novel therapeutic targets for patients with retinal neovascular diseases, an alternative to anti-VEGF therapy.


Social isolation has affected a large number of people and may lead to impairment of physical and mental health. Although stress resulting from social isolation may increase cancer progression, its interference on tumorigenesis is poorly known. In this study, we used a preclinical model to evaluate the effects of social isolation stress on chemically induced oral carcinogenesis. Sixty-two 21-day-old male Wistar rats were divided into isolated and grouped groups. After 90 days of age, the rats from both groups underwent oral carcinogenesis with 4-nitroquinoline 1-oxide (4NQO) for 20 weeks. All rats were assessed for depressive-like behavior and euthanized for oral squamous cell carcinoma (OSCC) diagnosis and measurement of inflammatory mediators in the tumor microenvironment. Social isolation stress increased the OSCC occurrence by 20.4% when compared to control. Isolated rats also showed higher tumor volume and cachexia than the grouped rats. Social isolation did not induce changes in the depressive-like behavior after carcinogenic induction. Tumors from stressed rats had increased levels of the inflammatory mediators, TNF-alpha, IL1-beta and MCP-1. The concentrations of TNF-alpha and MCP-1 were significantly increased in the large tumors from isolated animals. Higher tumor levels of TNF-alpha, IL-6, IL1-beta and MCP-1 were positively correlated with OSCC growth. This study provides the first evidence that social isolation stress may facilitate OSCC occurrence and tumor progression, an event accompanied by increased local levels of inflammatory mediators. Currently, several people worldwide are isolating during the COVID-19 pandemic –5. PeoplA growing number of studies have been investigating the association between emotional disorders and cancer progression –10. SociOral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer and occurs frequently in elderly men ,24. The ad libitum access to drinking water and standard pellet diet  throughout out the experimental period.All experiments with animals followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Welfare Committee at the São Paulo State University (UNESP), School of Dentistry, Araçatuba, São Paulo. Sixty-two male Wistar rats were obtained from the Central Animal Care Facility of the university and housed in ventilated cages, which were placed in an environment room with controlled temperature (25±2°C), humidity (55±5%) and light (12-h light/dark cycle). The animals had The study was conducted with two experimental groups: 1) Isolated group: 30 rats subjected to social isolation stress ; 2) Grouped group: 32 rats kept in groups of four animals per cage. After reaching 90 days of age, rats from both groups were subjected to oral carcinogenesis for 20 weeks. The animals underwent to behavioral tests one week before the end of carcinogenic induction. Histopathological analysis was performed to assess OSCC occurrence and tumor invasion. Levels of pro-inflammatory cytokines TNF-alpha, IL-6 and IL1-beta and the monocyte chemoattractant protein-1 (MCP-1) concentrations were analyzed in the OSCC microenvironment. st postnatal day, the rats from isolated group were separated from their mothers. This age was chosen to initiate isolation because it is critical for the development of social behavior . Le. Le38]. Correlation tests were performed in order to evaluate whether the concentrations of inflammatory mediators in the OSCC microenvironment were associated to the tumor volume . The levThe current study promotes the first evidence that social isolation stress may accelerate the onset and progression of OSCC in rats subjected to chemically induced carcinogenesis. Isolated rats displayed cachexia and increased levels of inflammatory mediators in the tumor microenvironment. Social isolation stress has been associated to tumor progression in preclinical models of liver  and breaOur results demonstrated that isolated rats had higher OSCC occurrence induced by 4NQO than the grouped animals. This carcinogen can promote the development of a variable number of lesions in oral cavity, oropharynx and esophagus. Our study found carcinogen-induced lesions only in dorso-posterior region of the tongue. No lesions were found in other regions of the oral cavity or in oropharynx and esophagus. So far, only one study has evaluated the impact of chronic stress on oral carcinogenesis in animals . In ratsAnimals subjected to social isolation since weaning may display a heightened stress response accompanied by the increased release of corticosterone in the serum . This upIncreased depressive-like behavior is one of the main consequences of chronic stress derived from social isolation –52. In tChronic stress may promote cancer progression through an enhanced tissue expression of tumor progression-related molecules . In the This study reveals for the first time that social isolation stress may increase OSCC occurrence in a pre-clinical model. Social isolation stress was also associated to an increased tumor growth, body weight loss and increased levels of inflammatory mediators in the tumor microenvironment.


Acute ischemic stroke treatment faces an unresolved obstacle as capillary reperfusion remains insufficient after thrombolysis and thrombectomy causing neuronal damage and poor prognosis. Hypoxia-induced capillary constriction is mediated by actomyosin contraction in precapillary smooth muscle cells (SMCs) therefore smooth muscle myosin-2 could be an ideal target with potentially high impact on reperfusion of capillaries.Methods: The myosin-2 inhibitor para-aminoblebbistatin (AmBleb) was tested on isolated human and rat arterioles to assess the effect of AmBleb on vasodilatation. Transient middle cerebral artery occlusion (MCAO) was performed on 38 male Wistar rats followed by local administration of AmBleb into the ischemic brain area. Development of brain edema and changes in cerebrovascular blood flow were assessed using MRI and SPECT. We also tested the neurological deficit scores and locomotor asymmetry of the animals for 3 weeks after the MCAO operation.Results: Our results demonstrate that AmBleb could achieve full relaxation of isolated cerebral arterioles. In living animals AmBleb recovered cerebral blood flow in 32 out of the 65 affected functional brain areas in MCAO operated rats, whereas only 8 out of the 67 affected areas were recovered in the control animals. Animals treated with AmBleb also showed significantly improved general and focal deficit scores in neurological functional tests and showed significantly ameliorated locomotor asymmetry.Conclusion: Direct inhibition of smooth muscle myosin by AmBleb in pre-capillary SMCs significantly contribute to the improvement of cerebral blood reperfusion and brain functions suggesting that smooth muscle myosin inhibition may have promising potential in stroke therapies as a follow-up treatment of physical or chemical removal of the occluding thrombus. Moreover, hypoxia is followed by the decline in ATP levels, which hinders the detachment of myosin from actin thereby populating myosin in the strongly actin-bound, rigor state. In addition, the formation of oxygen and nitrogen radicals during ischemia also contributes to pericyte constriction Restriction of blood supply during ischemic stroke was demonstrated to trigger the constriction of smooth muscle actin expressing contractile SMCs followed by SMC death in rigor These events lead to the irreversible contraction of brain capillaries resulting in prolonged decrease in blood flow, downstream thrombus formation and the concomitant damages of neurons.The most promising advances in regeneration after stroke have been achieved by influencing the Rho-associated kinase (ROCK) pathway. ROCK is a central hub of several different pathways regulating protein synthesis, cell growth, cytoskeleton rearrangements and acto-myosin contraction in non-muscle cell cortices para-aminoblebbistatin (AmBleb) We hypothesized that direct inhibition of SMM in pre-capillary SMCs might achieve the same positive effects as ROCK inhibition while avoiding the unwanted negative side effects of inhibiting an upstream hub regulator protein. In this study, we demonstrate that direct inhibition of SMM with a biologically safe blebbistatin derivative 99mTc]-HMPAO Brain-SPECT® kit was purchased from Medi-Radiopharma Co., Ltd. (Hungary). StemPro™ Neural Stem Cells and the required chemicals for cells were purchased from Thermo Fisher Scientific (USA). The isoflurane was purchased from Medicus Partner Ltd. (Hungary).Chemicals were purchased from Sigma-Aldrich (Germany) if otherwise not stated. -HMPAO  was administered to the animals 60 minutes prior start of SPECT imaging, via tail vein bolus injection in 300-600 µl volumes. The applied radiopharmaceutical, 99mTc--hydroxy-methylene-paraamino-oxime ([99mTc]-HMPAO) accumulates in brain tissue  as a function of the tissue blood perfusion in the CNS. After delivery by capillaries, the radiotracer is entrapped inside the cells behind the blood-brain barrier (BBB) and its quantifiable radioactivity is proportional to the initial perfusion of the given brain region.SPECT and MRI were carried out on rats anesthetized with 2% isoflurane in medical oxygen immobilized and fixed in a specifically designed animal anesthesia, restraining and heating bed . For SPECT detection of blood perfusion changes, 80-160 MBq [The SPECT measurements were performed on a NanoSPECT/CT+  imaging system. A helical data acquisition was performed in 50 minutes on each animal with 100 frames and 120 seconds per frame in a 256x256 column/row matrix with 125x125 μm/pixel resolution. Collected data were reconstructed using the Mediso Tera-TomoTM Monte-Carlo model-based maximum likelihood expectation maximization (MLEM) SPECT reconstruction algorithm running on the Nucline  software. Brain volumetric radioactivity data in 330 µm isovoxels were obtained and quantified in units of radioactivity measured in MBq/ml.99mTc]-HMPAO injection, using a nanoScan® 1T MRI system  with a dedicated transmit/receive rat coil. A T2 weighted Fast Spin Echo (FSE) three-dimensional sequence had 460 µm in-plane resolution, slice thickness: 460 µm, repetition and echo times 2000 ms and 78.9 ms, 80 frequency- and 64 phase encodings, 90 degrees flip angle and 2 excitations resulting in a 25 minutes 44 seconds acquisition. Image analysis of 3D SPECT and MRI VOIs was performed with VivoQuant software . MRI images were employed as reference to the SPECT of the same animal in each imaging time point. During analysis, each animal's SPECT/MRI co-registered image was also co-registered to a MRI brain atlas template using the co-registration tool in SPM12 (7219).MRI acquisitions were performed immediately following the [Firstly, to quantify the MRI and SPECT recordings, the extrameningeal tissues were discarded. Using BrainSuite18a  the skull tissues were removed from the MRI volumes using the skull-stripping pipeline with additional manual alignments in each slice. The final brain masks were applied to the previously co-registered SPECT volumes as well.For the quantification of the cerebral edema volume on the MRI dataset, the pixel intensity histograms of the hemispheres were determined using the stack histogram tool in Fiji 99mTc-HMPAO dataset of healthy rats A parametric map was created from our masked SPECT recordings and the SPECT The co-registered parametric maps were imported to MATLAB R2018b for further analysis:1. The mean ± SD values of the physiologically normal relative rCBF for each functional brain region were calculated from the dataset of healthy rats. The hypo- and hyperperfused voxels of MCAO treated rats were selected by setting up a threshold for both by subtracting/adding 2xSD to the mean rCBF values of the reference dataset, corresponding to the 95% confidence interval in each individual brain region. Hypo- and hyperperfused brain volumes were constructed by grouping voxels outside these thresholds. 2. We evaluated the relative rCBF changes over time by using the day 0 hypoperfused volume as a 3D mask. The mask was applied to all measurements in the time-series of the same animal and either the voxels contained in these ROIs of the parametric maps were averaged for each time point (voxel-wise), or the SPECT signals were averaged and then transformed using Lassen's algorithm (roi wise). 3. The relative rCBF calculation for the functional regions present in the rat brain atlas was performed for each measurement point by averaging the voxel values corresponding to each region. By relating the values to those of the reference dataset, we obtained discrete relative rCBF values for every functional region. We classified brain regions into two groups based on whether the rCBF value was significantly different (non-regenerated) from the reference regions or not (regenerated).To avoid abusing the animals with imaging procedures and neurological test assessments on the same day, neurological scoring tests were carried out 1 day after the imaging days except for day 21. The neurological status of the animals was assessed on days 4, 8, 11, 15, 18 and 21 after MCAO by scoring tests relating to focal and general deficits In case of general deficits animals were scored between 0-4 or 0-2 based on the severity as follows: posture , spontaneous activity , hair  and eyes . Since the condition of eyes was almost completely recovered at day 8 in both control and treated groups therefore it was excluded from the cumulative analysis of general deficit scoring.In case of focal deficits animals were scored between 0-4 based on the symptoms as follows: body symmetry , gait , front limb symmetry , compulsory circling  and Whisker-response . For analysis, average scores ± SEM were calculated at each time point for the control and treated groups, significance of difference was evaluated by unpaired t-test.To evaluate locomotor asymmetry, MCAO treated rats were tested in an open-top, 21 cm diameter, clear glass cylinder 4, 8, 11, 15, 18 and 21 days after MCAO operation and AmBleb treatment. Forelimb activity was calculated from number of touches of each forelimb against the wall of the arena. Forelimb use was defined as the whole palm touched the sidewall of the cylinder. Number of sidewall touches was counted for 2 minutes.The number of replicates (n) is indicated in the figure legends and refers to the number of experimental subjects independently treated in each experimental condition. Data are presented as means ± SEM. Statistical analysis was carried out using unpaired two sample t-test in GraphPad Prism 7 and normality was tested using the Shapiro-Wilk test unless otherwise stated. Statistical significance was set at *p < 0.5, **p < 0.01.in vivo. We measured the vasodilatation effect of AmBleb on isolated rat and human arterioles (Figure ,in vitro results encouraged us to investigate the effects of AmBleb in in vivo rodent stroke models.Blebbistatin has been previously reported to be an inhibitor of SMM and arterial contractions Figure ,. ContracFigure ,D-E. ImpoFigure ,E. These As a proof-of-concept for the positive effects of direct myosin-2 inhibition on recovery after stroke, MCAO was applied to model acute ischemic stroke on 34 male Wistar rats. To maximize reproducibility we did not include females, as sex may influence the response to neuroprotective agents We also investigated the effect of AmBleb treatment on cardiovascular and respiratory functions . We admiBrain tissue edema was identified on the skull-stripped MRI images based on the intensity histograms of the two hemispheres. In agreement with other studies We followed the change of the total hypoperfused volume in the treated and untreated animals. After 3 days of MCAO, a striking difference was observed between the control and the AmBleb treated animals and the difference remained significant for another 11 days Figure F. A sligWe also determined the changes in rCBF values in the initially hypoperfused region for 21 days (Figure From the total number of voxels that are significantly different from the healthy counterparts we could define the stroke-affected brain volume as a function of distance from the injection site Figure H. These By day 7 in the ipsilesional hemisphere the hypoperfused volume shrank by 67% in the treated, while only 1% in the untreated animals. In the contralesional hemisphere the volume of the hypo- and hyperperfused areas did not change significantly in the treated and the untreated animals indicating direct local effect of AmBleb Figure .In healthy animals there are both characteristic differences in rCBF levels between the functional regions within a hemisphere, and also region-specific differences between the two hemispheres Figure . To idenAfter MCAO the general and focal neurological deficits were followed on 16 control and 15 AmBleb treated animals including the ones tested in MRI/SPECT imaging (Figure We further analyzed focal deficits of the animals in body symmetry, gait, front limb symmetry, compulsory circling and Whisker response Figure B. Cumulain vivo experiments, and AmBleb treatment significantly enhanced the rCBF and accelerated neurological improvements after MCAO in the 3-week follow-up period. The observed changes in rCBF values and neurological scores of the control group are in line with previous studies We demonstrated that AmBleb has drastic effect on vasodilation of both rat and human arterioles due to the effective relaxation of SMM in the same concentration range as the inhibitory constant for myosin-2 ATPase activities. We propose that the effective vasodilatator effect is realized in the Although AmBleb is not myosin-2 isoform specific, the locally applied effective dose is more than 100-times less than the dose administered systematically with no effect on cardiovascular and respiratory functions , suggestOur advance in improving recovery after stroke by direct SMM inhibition provides a new concept that earlier successes in MCAO post-stroke regeneration with ROCK pathway inhibitors Direct myosin-2 inhibition could also affect other substantial processes during stroke regeneration, which might also contribute to improved rCBF values after AmBleb treatment. It was demonstrated that myosin-2 inhibition promotes angiogenic sprouting and initial vascular branching in vivo results provide a proof-of-concept and encourage the development of a translationally more relevant administration of the drug and further tests on more complex disease models, such as animals with cardiovascular diseases. In addition, the combination of AmBleb with edema reducing therapies could have the potential to extend the efficiency of the treatment for ischemic stroke and hence improve the neurological outcomes The presented We established an unbiased method to assess the effects of MCAO and the dynamics of recovery in a large number of hypoxia-affected brain regions using published reference values of healthy rats. By using an independent reference set instead of comparing the ipsilesional and contralesional hemispheres, we were able to define the affected regions on both hemispheres after MCAO and found substantial improvement in the ipsilesional side of the AmBleb treated group. As previously shown, ischemia-induced hypoperfusion may affect certain brain regions differently based on the cerebral blood flow reduction, the degree of collateral circulation and the metabolic activity in human patients We also showed that direct targeting of the effector molecule, SMM, has the same positive consequence but without the possible broad side-effect profile emerging from the inhibition of a central regulatory hub kinase, ROCK. The effects of the pharmacologically safe myosin-2 inhibitor, AmBleb indicate that SMM is a promising drug target candidate in ischemic stroke treatment. Thus, further studies are encouraged to assess the effect of myosin-2 inhibition on regeneration after stroke involving fine sensorimotor and cognitive tests on older animals especially with comorbidities to more prudentially approach the human risk conditions according to the current consensus-based guidelines in stroke research Direct targeting of SMM in precapillary SMCs and consequent opening of permanently blocked capillaries in the ischemic brain regions may fulfill both short- and long-term requirements towards an innovative medication. In addition, its combination with the current treatments has the potential to extend the treatment-eligibility criteria thereby allowing effective therapies to a broader population of stroke patients.Supplementary figures and tables.Click here for additional data file.


The Journal of Innovations in Cardiac Rhythm Management by Boey et al.1 reviews sudden cardiac death (SCD) risk prevention for patients with structural heart disease and impaired left ventricular function. Patients with inherited channelopathies ; genetic disorders such as arrhythmogenic right ventricular dysplasia (ARVD) or hypertrophic cardiomyopathy (HCM); and those with unique statuses such as having cardiac sarcoidosis or awaiting heart transplantation are not reviewed. Unfortunately, the lack of prospective randomized trials for these conditions means they will likely never reach class I or IIa indications for implantable cardioverter-defibrillator (ICD) implantation, despite the obvious potential benefit to patient survival.The manuscript in this issue of 2 raises important questions and perhaps the largest benefit for nonischemic cardiomyopathy subjects with left bundle branch block and QRS duration > 120 ms is from cardiac resynchronization therapy (CRT) pacing rather than the presence of a defibrillation lead. A randomized trial comparing the use of CRT-pacemakers and CRT-defibrillators would be greatly welcome in this population, in particular in elderly patients.Prospective randomized clinical trials for ICD implantation for secondary prevention have firmly established the role of the ICD in preventing recurrent sudden death. Primary prevention ICD trials involving high-risk subjects post-myocardial infarction who have a left ventricular ejection fraction (LVEF) < 30% for more than 40 days or an ischemic LVEF < 35% and class II/III systolic heart failure are similarly strongly supported. As the authors nicely review, the benefit of primary prevention ICD implantation in the nonischemic cardiomyopathy patient has been questioned. The DANISH ICD trial3) and supports the expanding consideration of covering the high-risk patient with a LifeVest  or a “wearable defibrillator.” Deferring the decision to implant a permanent ICD until medical therapy has been optimized and the subject has proved the ability to survive the early at-risk period is an evolving option needing further study. The majority of LifeVest  use at Vanderbilt Medical Center is post-lead extraction in patients awaiting reimplantation. However, the rapid growth of our ventricular assist device and heart transplant program has expanded routine LifeVest  use to early on while awaiting heart transplant evaluation in some patients, avoiding the risk of invasive ICD implantation altogether.Early implantation of an ICD has not proven effective at reducing overall mortality  may reduce the potential risks of lead dislodgement, pneumothorax, and fracture, these devices are often implanted in the worst clinical candidates. Patients with prior cardiac implantable electronic device infections have a high one-year mortality and those with end-stage renal disease have not demonstrated any benefit from ICD therapy. Importantly, these are often the patients S-ICD implantation is first considered in, thus limiting the potential widespread benefit of the therapy. Additionally, implantations are typically performed under general anesthesia, adding some cognitive risk. S-ICDs can be prone to undersensing when liberties are taken with screening electrocardiograms to determine adequate QRS and T-wave morphology in order to quality a patient for device implantation, or when they are implanted in obese subjects. An unintended benefit of the delayed S-ICD detection and charge time to reach 65 Joules (J) or 80 J is the allowance for more sustained ventricular arrhythmias to prove themselves to be “non-sustained” by spontaneously converting prior to ICD shock. Though the youngest patients may derive the greatest long-term benefit from S-ICD implantation, this is also generally the population of patients with hypertrophic cardiomyopathy, long QT syndrome, or ARVD who are prone to inappropriate sensing and/or T-wave double-counting and who often benefit from the presence of antitachycardia or atrial pacing support.4 and VTACH5 trials.Finally, when considering the use of an electrophysiology study (EPS) to induce ventricular tachycardia (VT) or ventricular fibrillation (VF) for the placement of a primary prevention ICD in patients with an LVEF > 35%, the devil is in the details. There is no specific, uniform induction sequence for the EPS and, depending on the operator’s desire to implant a device, the protocol can be tailored to either increase or decrease the likelihood of inducing VT, VF or ventricular flutter. VF is not primarily a reentrant arrhythmia and, with the progress and efficacy of VT ablation, patients with induced VT in the electrophysiology laboratory may be better suited for VT ablation and implantable loop recorder monitoring for spontaneous events. This would be fertile ground as well for a randomized trial in the future on the tail of the SMASH-VT


Diabetic retinopathy is a vision-threatening disease affecting neurons and microvasculature of the retina. The development of this disease is associated with the action of inflammatory factors that are connected to the activation of microglial cells, the resident tissue macrophages of the CNS. In the quiescent state, microglial cells help maintain tissue homeostasis in the retina through phagocytosis and control of low-grade inflammation. However, prolonged tissue stress due to hyperglycemia primes microglia to become overly reactive with the concomitant production of pro-inflammatory cytokines and chemokines causing chronic inflammation. In this review, we provide evidence of microglial cell activation and pro-inflammatory molecules associated with the development and progression of diabetic retinopathy. We further highlight innovative animal models that can mimic the disease in humans and discuss strategies in modulating microglial-mediated inflammation as potential therapeutic approaches in managing the disease. Diabetic Retinopathy (DR) is a disease that affects the neurons and microvasculature of the retina leading to vision deficits in people suffering from diabetes mellitus (DM). DR is clinically divided into two broad forms based on the severity of the disease; an early non-proliferative form (NPDR) and an advanced proliferative (PDR) form. NPDR is characterized by increased vascular permeability, capillary occlusion, retinal hemorrhages, hard exudates, thickening of the basement membrane, and pericyte drop-out in the retinal vessels. In contrast, PDR entails pathological neoangiogenesis, vitreous hemorrhage and retinal scars and detachment . A critiDR was initially regarded as a microvasculopathy owing to the abnormalities in the retinal microvasculature such as microaneurysms and acellular capillaries . HoweverMicroglia are mononuclear phagocytes and can be regarded as the tissue-resident macrophages of the retina. There has been a debate regarding their origin but it is now generally accepted that they originate from the embryonic yolk sac and are self-renewing . The yolIn early stages of retinal development, microglia inhabit the ganglion cell layer of the retina and are involved in phagocytosis of dying neurons . In the These glial cells not only possess the protective ability but also play a part in the development and preservation of neuronal circuits. In healthy situations, microglia through their receptors for neurotransmitters establish molecular links with neurons and other cells through which they participate in the maintenance of neural networks and the removal of cell fragments through phagocytosis –23. It ilo, CD11clo, F4/80lo signature which is conserved in steady-state and disease while monocyte-derived macrophages express the same markers in higher quantities  , 40. DAMignature . During ignature  where thignature . In viruignature .Several animal models have been used to study the activation of microglia in diabetes and have confirmed the phenotypic changes mentioned herein. In the streptozotocin (STZ)-induced diabetic rat, microglia change their morphology from a ramified to an amoeboid form and increase in number within the outer plexiform layers and outer photoreceptor layer suggesting that activated microglia are highly proliferative and migratory . In anotIn the mouse model of oxygen-induced retinopathy (OIR), resident microglia with a proliferative and migratory phenotype were found to occupy ischemic and neovascular regions of the retina . FurtherElevated glucose levels and glucose fluctuations can induce microglia polarization in cell culture systems. The treatment of rat microglia with increasing glucose concentrations upregulated expression of TNF-α and monocyte chemotactic protein-1  in a time dependent manner . A studyAt the onset of DR, the BRB offers a physical barrier that prevents circulating cells from entering the retina and this leaves microglia and other retinal cells as the only immune sentinels of the retina. In this state, the low-level tissue insults are detected by microglia through their pattern recognition receptors (PRRs). Microglia maintain several immune checkpoints to restore tissue homeostasis. In the presence of an early noxious stimuli such as hyperglycemia, microglia maintain a balance of the pro- and anti-inflammatory states but as the tissue insults persist the balance is lost and the cells are reprogrammed to attain a new threshold characterized by the pro-inflammatory state which is harmful  and nuclear factor kappa B (NF-kB)  pathways. This phenomenon results in increased production of reactive oxygen species (ROS) and accumulation of advanced glycation or lipoxidation end-products (AGE or ALEs) which affect retinal physiology . Interac (NF-kB) . The hypnd TLR-4 , 64, thend TLR-4 . This glnd TLR-4 , 67.–/–Vldlr mice  translocation to the nucleus and ERK1/2-NF-kB signaling pathway in microglia , 69. Ove–/– mice . Conside–/– mice . The gly–/– mice . Increas–/– mice , 73. Mic–/– mice . Taken tThe initial inflammatory response of microglia is corrective and involves the release of anti-inflammatory cytokines including IL-4, IL-10, IL-13, and transforming growth factor-beta (TGF-β) that resolve inflammation and enhance survival of neurons . Howeverβ, IL-6, and TNF-α) can induce the secretion of C1r, C1s, and C3 by astrocytes and microglia  signaling is important for the complete formation of the functional BRB , db/db (Leprdb), Kimba, and Akimba. The Ins2Akita and NOD mice were developed to study type 1 diabetes whereas the db/db (Leprdb) mice were developed as models for type 2 diabetes. The Akimba mouse was generated by crossing the non-diabetic Kimba mouse model of proliferative retinopathy with diabetic Ins2Akita mouse  or genetic mutation (breeding) to investigate the pathophysiology of DR, and to design and assess novel therapies for DR. Disease phenotypes in STZ diabetic mice and rats include increased astrocyte gliosis, thinning of inner and outer retinal nuclear layers, pericyte ghosts, BRB breakdown, acellular capillaries, and basement membrane thickening . Geneticta mouse . Single ta mouse . AlthougPdgfbiΔEC mouse model generated by deleting platelet-derived growth factor (PDGF)-B in retinal endothelial cells (ECs) reproduced the key features of DR  research unit FOR2240 (project 6) and the CMMC Cologne (project B05).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To evaluate the Effectiveness of Pre-operative Respiratory Muscle Training versus Conventional Treatment for Improving post-operative pulmonary health after Coronary Artery Bypass Graft Surgery (CABG).A Prospective Randomized clinical trial was performed on sixty patients who underwent elective CABG at Faisalabad Institute of Cardiology. At the time of admission all patients were subjected to 6-minutes’ walk test (6MWT) as baseline. The subjects were then divided into two groups. The Group-I was subjected to respiratory muscle training whereas the Group-2 received the routine preoperative care. The 6-minute walk test (6MWT) was then repeated a day before surgery (pre-operative) and before discharge (post-operatively). Duration of post-operative mechanical ventilation, oxygen therapy and hospital stay were also noted as outcome measures of this study.The pre-operative and post-operative readings showed that the patients in the interventional group performed better than the control group in their 6MWT with P-value of less than 0.05. Similarly the interventional group had shorter duration of mechanical ventilation, dependence on oxygen therapy and postoperative hospital stay as compared with the control group showing P-values below 0.05.The results showed that respiratory muscle training results in improved postoperative functional capacity and reduces of hospital stay. The available literature supports that one of the major cause of post-operative pulmonary problems which increases the duration of mechanical ventilation and oxygen therapy was poor preoperative pulmonary health. Therefore, there is need to find if any improvement of pre-operative pulmonary health in patients selected for Coronary Artery Bypass Grafting has impact on outcome of surgery. The purpose of this study was to evaluate the effectiveness of preoperative inspiratory muscle training in patients undergoing Coronary Artery Bypass Grafting and assess their pulmonary status after surgery.Both male or female patients within age group of 40-65 years planned for coronary artery bypass grafting and who were willing to participate in the study were included. The patients showing inconsistency with initial stages of exercise rehabilitation were excluded. Similarly patients suffering from asthma, COPD, interstitial lung disease, endocrine abnormalities such as hyper or hypothyroidism, neurological disability affecting respiratory rate, left ventricular ejection fraction <40%, concomitant valve disease, dysrhythmias, pacemaker dependency, anemia and obesity were also excluded from the study.A total of 60 patients were included in study and they were randomized to Group-I as Intervention Group and Group-2 as Control group. The mean age in group Group-I was 53.97 years with standard deviation of 7.29 years while the mean age in Group-2 was 54.13 years with standard deviation of 4.17 years. The difference between the mean age of both groups was not significant (P=0.91). The gender distribution in both groups was slightly disproportionate. The Group-I had 27 male and three female patients whereas the Group-2 had 22 male and eight female patients. However, the difference in gender distribution of both groups was not significant as the p-value calculated by chi-square test was 0.09.“There is no significant difference between respiratory muscle training and conventional treatment”.“There was significant difference between respiratory muscle training and conventional treatment”.Modified Healthy Heart Questionnaire (HHQ-GP-1) was used as a screening tool and The 6MWT was performed as a baseline in Group-I and Group-2 on the day of admission. The distance covered (feet), heart rate and oxygen saturation were noted after the 6-minutes walk. Portable finger Pulse Oximeter was used for assessment of oxygen saturation and heart rate. After this the patients in the Group-I received respiratory muscle training (RMT) which consisted of the incentive spirometer (IS), diaphragmatic breathing, segmental breathing movement  and huff-coughing techniques. A total of 28 sessions of respiratory muscle training were held in Group-I for >15 minutes at a constant low speed for each patient. The patients in Group-2, received conventional treatment which includes deep breathing. The above mentioned 6-minute walk test was then repeated a day before the surgery  and then at the time of discharge after surgery .Statistical analysis was performed by using SPSS version 20. Independent Sample T test was applied on numeric data. The difference in the gender distribution was evaluated by Chi-square test. The P-value of 0.05 or below was considered as significant.The results showed that there was significant difference between interventional and control group in their distance covered in six minute walk test, oxygen saturations and heart rate. Similarly duration of mechanical ventilation, dependence on oxygen therapy and the length of post-operative hospital stay were also statistically different in both groups.The The arterial oxygen saturation levels are known to improve after inspiratory muscle exercise pre-operatively and before discharge.Elkins et al have reported that inspiratory muscle training significantly improved rapid shallow breathing.8There are several reports regarding the efficacy of perioperative breathing and coughing exercises in prevention of post of pulmonary complications (PPCs). Stiller et al, 1994) investigated the efficacy of 3 to 5 deep-breathing and coughing exercise in the prevention of PPCs after CABG and reported that prophylactic chest physical therapy did not prevent PPCs after CABG.94 invest11The finding of the present study was in accordance with Matheus et al, who showed a substantial reduction in length of stay in the coronary care unit in the study group and present study also indicated reduction in days of ICU stay.13It is concluded that inspiratory muscle training is effective in improving the respiratory muscle strength and functional capacity of patients elected for cardiac surgery. This training results in better pulmonary outcomes after CABG.WS: Project concept; Data collection, Statistical Analysis and Manuscript writingNA: Manuscript writingZH: Study DesignAJ: Supervision; Manuscript EditingWS takes the overall responsibility and is accountable for ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.


Astrocytes are specialized glia, vital for neural circuit function, and represent a population of complex and functionally diversified cells . PhysiolThus research has the challenge to pharmacologically regulate astrocyte functions with special focus on reducing neural aberrant excitation and promoting restorative signals.The present research topic is intended to be a collection of new physiological and pathological evidence regarding astrocyte features and functions focusing on the concept that astrocytes represent a highly variegated population of cells that mediate neural circuit-specific roles in health and disease.Spampinato et al. have focused on two important astrocyte functions with pathophysiological relevance: i) regulation of neural stem cell properties within adult neurogenic niches, positive pleiotropic actions of utmost importance under neurodegenerative conditions as an attempt to replace lost cell populations and ii) regulation of the integrity and functions of the blood-brain barrier (BBB) in physiological condition and as a reaction to harmful events contributing to either exacerbate or reduce BBB damage.Valenza et al., it is mainly expressed on astrocytic endfeet closest to blood vessels participating in several astrocyte signals. The review points out the latest AQP-4 findings related to aging and Alzheimer’s disease as well as the available knowledge on pharmacological tools to target AQP-4.Another crucial physiological need satisfied by astrocytes is the cleansing of the cerebral tissue from waste molecules. Aquaporin-4 (AQP-4), a brain water channel, plays a pivotal role in this process. As shown in the review article of Venturini et al. have found that astroglial processes could release neurogloblin-containing exosomes as new non-conventional signals.As regards the cross-talk with the other nervous cells, classic astrocyte-to-neuron communication encompasses the release of messengers via exocytosis, carrier membrane transport and opening of a wide-range of channels . NeverthLagos-Cabré et al. have reviewed and discussed evidence suggesting that cell adhesion and cytoskeletal dynamics, both of which are relevant to cell migration, take place by modulation of hemichannels rather than gap junction channels.Among channels implied in the intercellular crosstalk, connexins  represenLozzi and co-workers, by using bioinformatic approaches have demonstrated that cohorts of transcription factors may modulate region-specific molecular signatures in astrocytes. This evidence points out the idea that differential expression of transcription factors governs astrocyte diversity in the brain parenchyma.Despite the evident astrocyte complexity in terms of phenotype and function, the molecular basis of these differences are unclear. Chen and co-workers showed the protective action of melatonin from heme-induced toxicity observed upon intracerebral hemorrhage. They found that this response is mediated by the activation of M2 receptors and the transcription factor Nrf2.Melatonin is produced in the pineal gland and released according to the circadian rhythm . RecentlHostenbach et al. determined that diversity of pro-inflammatory cytokines causes the production of ET-1, the latter being dramatically prevented by the statin and the natural phenol simvastatin and resveratrol, respectively.Astrocytes do not express endothelin-1 (ET-1) in healthy conditions, but they prominently express and release this protein in multiple sclerosis demyelinated plaques . In thisSiracusa et al. The loss of astrocyte functionality as a result of cellular senescence has been related to neurodegenerative disorders as well as to aging. Astrocytes can drive the inflammatory response and contribute to the altered neuronal activity in several frontal cortex pathologies such as ischemic stroke and epilepsy. For these reasons, the authors discuss the possibilities to target astrocytes as an approach toward pharmacological therapies.The relevance of astrocytes in pathological conditions was deepened by Zhao et al., vinpocetin, a semi-synthetic alkaloid from the leaves of Phyllostachys pubescens, has anti-inflammatory, anti-oxidant and anti-apoptotic actions both in vitro following oxygen-glucose deprivation and in vivo against ischemia/reperfusion injury by targeting specific astrocytic pathways. Specifically, it promotes Connexin43 phosphorylation through the PI3K/Akt pathway, which in turn promotes BBB integrity, cell-to-cell communication with an overall reduction in brain edema and tissue damage.In this view, astroglia is implicated in the pharmacodynamic of already known products. Recent developments have demonstrated that astrocytes can indeed be the cellular targets of neuroprotective agents. As demonstrated in the paper by Liparis nervosa (Thunb.) Lindl. has been studied by Liu et al. in an in vitro model of oxygen-glucose deprivation/reoxygenation injury (OGD/RI) on astrocytic cultures to mimic the pathological condition named neonatal hypoxic/ischemic. Data demonstrate that TTB is effective against cell death preserving the intracellular antioxidant activity by activating the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and related pathways. Additionally, TTB reverts neurite loss induced by OGD/RI in neuron-astrocyte cocultures.The natural compound 2,7,2′-trihydroxy-4,4′7′-trimethoxy-1,1′-biphenanthrene (TTB) isolated from the orchid In conclusion, this Research Topic offers novel information about the role of astrocytes in neurophysiology and in neuropathology as well as possible therapeutic approaches. The pharmacological modulation of astrocytic targets is encouraged as a breakthrough strategy for the relief from several debilitating pathologies.


Significant alterations of cambial activity might be expected due to climate warming, leading to growing season extension and higher growth rates especially in cold-limited forests. However, assessment of climate-change-driven trends in intra-annual wood formation suffers from the lack of direct observations with a timespan exceeding a few years. We used the Vaganov-Shashkin process-based model to: (i) simulate daily resolved numbers of cambial and differentiating cells; and (ii) develop chronologies of the onset and termination of specific phases of cambial phenology during 1961–2017. We also determined the dominant climatic factor limiting cambial activity for each day. To asses intra-annual model validity, we used 8 years of direct xylogenesis monitoring from the treeline region of the Krkonoše Mts. (Czechia). The model exhibits high validity in case of spring phenological phases and a seasonal dynamics of tracheid production, but its precision declines for estimates of autumn phenological phases and growing season duration. The simulations reveal an increasing trend in the number of tracheids produced by cambium each year by 0.42 cells/year. Spring phenological phases  show significant shifts toward earlier occurrence in the year (for 0.28–0.34 days/year). In addition, there is a significant increase in simulated growth rates during entire growing season associated with the intra-annual redistribution of the dominant climatic controls over cambial activity. Results suggest that higher growth rates at treeline are driven by (i) temperature-stimulated intensification of spring cambial kinetics, and (ii) decoupling of summer growth rates from the limiting effect of low summer temperature due to higher frequency of climatically optimal days. Our results highlight that the cambial kinetics stimulation by increasing spring and summer temperatures and shifting spring phenology determine the recent growth trends of treeline ecosystems. Redistribution of individual climatic factors controlling cambial activity during the growing season questions the temporal stability of climatic signal of cold forest chronologies under ongoing climate change. This vaNorthern and high-elevation forests experience prominent year-to-year changes in cambial dynamics (process of xylem tissue development), phenology  and kinetics (speed of xylem cell differentiation). The initiation of cambial activity in cold regions is driven mainly by temperature . ConsequThis lack of long-term data on trends in cambial dynamics is especially unwelcome in areas where forests have shown significant trends in tree growth. For instance, most stem increment chronologies from mountain treelines in Central Europe show significant increases in growth coupled with increasing temperatures . WithoutThe lack of long direct cambial dynamics observations can be overcome by application of climate-driven, process-based models of wood formation. They represent mathematical approaches that decompose annually resolved dendrochronological proxies  into intra-annually resolved growth components . The curIn this study, we aim to address the lack of validation of intra-annual performance of process-based models and assess the importance of intra-annual growth rates and phenology for annual growth trends. We used the Vaganov-Shashkin model (further abbreviated VS-model), which is a sink-oriented process-based model applicable to conifers, simulating kinetics  of cambium and its derivatives aligned in a radial cell file purely from daily resolved climatic data . We focuPicea abies (L) Karst.), has an upper margin situated at elevations 1290–1450 m  cross-dated using a TimeTable measurement stage  and PAST4 software . One core from each tree was extracted using Pressler corer at the height of 1 m from the stem base. All cores were mounted on wooden slices and sanded using progressively finer sandpapers to enhance visibility of tree-ring borders. Tree-ring widths were measured and visually and statistically  took place during the 2010–2017 growing seasons , 2019. TThe laboratory processing of microcores followed standard wood anatomical protocol  to produr = 0.97–0.98 for individual months), and their precipitation was also highly significantly correlated (r = 0.61–0.89).We obtained daily resolved climatic data  for the Szrenica climate station, located approximately 10 km from our sampling sites in a similar mountain environment . Data from this station are available for the years 1961–2001. For the period 2001–2017 we used data from the Labská bouda station, which is located nearby . Data from Labská bouda were corrected using parameters of linear regression calculated over the period of simultaneous measurement for both stations (1979–1999). Correlations indicate that there was virtually no difference in temperature between those stations ] and moisture [GrM]. Partial growth responses represent climatically driven growth potential on a scale from 0 to 1 (E(d)] to estimate a daily integral growth rate [Gr]  < GrM), moisture-limited  < GrT) or climatically optimal  = GrT = 1)  converts daily climatic conditions into a set of dimensionless indexes defining daily climatic potential for tree growth. Its inputs include site tree-ring width chronology , daily resolved climatic data  and daylength estimated from site latitude . A key cm 0 to 1 . The lowGr] . In addi,y) = 1) . Integra,y) = 1) .sensubeg) exceeds a certain threshold ; (ii) grows in diameter in “cambial zone”  > Vmin(j)); or (iii) loses ability to divide and starts differentiation to become a xylem tracheid by entering the “enlargement zone” (Vmin(j) > V > Vcr). If the cambial cell diameter exceeds certain threshold (YM), it enters the mitotic cycle and divides into two daughter cambial cells with equal diameter. It is important to note that the VS-model simulates only the process comprising cambial cell growth, division and occurrence of new differentiating cells (entrance into enlargement zone), not specific stages of tracheid differentiation and processes determining final tracheid anatomy . Consequently, it is not possible to determine daily numbers of cells in specific phases of differentiation , only the total number of cells that are in any of those stages (EWM). Simulated tree-ring width chronology is defined as the final annual simulated number of differentiated cells standardized by the mean number of differentiated cells per year over entire chronology period.In the next segment , the VS-model simulates cambial cell kinetics and differentiation of new tracheids aligned in a radial file . The onl1 threshold during the main growing season (delimited by DOYs 150–250), position-specific growth rates of cambial cells ) do not drop to 0 immediately, but only after acclimation period determined by the new parameter τ. In our case, τ = 3 days provided the best results when testing all possible values between 0 and 30 days. During the acclimation period, V of all cambial cells equal Vcr . This means, that cambial cells can sustain radial growth and undergo mitosis but are not permitted to enter differentiation zone during acclimation period. Thus, the growth processes are active only in the cambial zone and new enlarging tracheids do not appear during acclimation.Our initial exercise with original VS-model algorithm  indicateVaganov-Shashkin model is driven by a set of 10 parameters of the environmental block and 12 parameters of the cambial block; in addition, there are seven parameters of hydrological model simulating soil moisture. We used the standard methods to find or set their values . First, Independently, we verified estimated values of parameters by employing the differential evolution approach using a high-performance computer cluster. This approach finds values of a multidimensional domain of real-valued functions, which are optimal for a given environment . All valM and GrT for each day . Finally, we summed daily growth rates for dates with different types of climatic limitation and quantified their trends using linear regression.The following data were extracted from VS-model outputs: simulated tree-ring width chronology; cumulative annual growth rate during days with different climatic limitations; and simulated number of differentiating cells in radial file for each date of xylogenesis sampling, standardized by the final number of differentiated cells at the end of the year. In addition, we extracted four key dates describing the timing of cambial phenology and tree-ring formation for each year from the cambial block. As mentioned above, the VS-model estimates timing of cambial cell spring reactivation and autumn dormancy using the “heat-sum” approach . We alsoU test (random dispersion of simulations around observations). In addition, the date of the first/last occurrence of enlarging cell (observation) is mimicked by the date of the first/last entrance of cell to differentiation zone (simulation). We constructed scatterplots between the simulated and observed dates of the first and last appearance of differentiating cell and calculated their regression parameters. Temporal stability of model parameters and their independence on the range of calibration data was tested using a bootstrapped transfer stability tests  and Mann–Whitney ty tests  between sensup < 0.001) increasing trend in tree-ring widths since 1961, steepening during the most recent decades  and high soil moisture  on tree growth  did not drop below 0.87, whereas the partial growth response to temperature (GrT) peaked at 0.86 (DOY 213). The highest growth rate occurred between DOY 172 and 239, when mean GrT exceeds 0.6.On average, the growth at our study region was limited by low temperature during entire growing season . The meaT < GrM), whereas drought dominated in 22% (GrM < GrT). The remaining 35% of growth happened during optimal climatic conditions (GrM = GrT = 1).Considering daily model outputs, however, periods of various durations (from single days to months) with drought limitation or climatically optimal growth were identified in specific years. In total, 10773 days of cambial activity were simulated for the period 1961–2017 . Tempera–1 (≈0.8% of long-term mean). Similarly, total sum of integral growth rates over the entire growing season increased by 0.37 year–1 (≈0.5% of long-term mean), which was driven mainly by increasing growth in climatically optimal conditions , and residuals are randomly distributed according to Mann–Whitney U test for all years except 2010, 2011, and 2014 (2), but the similarity of variability (slope) slightly increases over time , 145 (±15), 277 (±17), and 308 (±15), respectively. There were negative temporal trends in cambial cell growth onset and appearance of the first differentiating tracheid, and positive trends in autumn cambial cell growth cessation and appearance of the last differentiating tracheid . HoweverThe results of this study demonstrate that combination of process-based models of wood formation and extensive observational data allows the identification of specific model limitations as well as extension of our understanding of processes modulating cambial activity on long temporal scales. Below, we frame implications of our results relevant for both methodology (limitations of the VS-model indicating the future development directions) and environment-growth interaction .The VS-model precisely predicted the date of the first differentiating tracheid appearance, for which mean model residuals are smaller than the frequency of xylogenesis sampling. Moreover, simulated and observed proportions of formed cells are tightly synchronized mainly at the beginning of the tree-ring . This isIndeed, VS-model does not consider any of non-climatic factors which might influence process of cell division and differentiation. The simulated progress of increasing number of cells was synchronized with xylogenesis data for five of eight considered years, while for other 3 years the model suggested delayed formation of the tree-ring. Consequently, simulated dates of the last differentiated cell appearance are unprecise. The termination of xylem formation is regulated partly by internal physiological processes, for instance, intra-annual patterns of hormone concentrations  or filliThe declining precision of VS-model through growing season resulting in biased estimate of growing season cessation was recently confirmed also in a boreal cold forests in Canada . SurprisP. abies and environmental conditions at our study site. The sensitivity analysis based on parameters of environmental block revealed, that variability in tree-ring widths is driven primarily by the effect of low temperatures and high soil moisture, which are common growth determinants at the treeline environment (1 = 5°C) is similar to values reported for most treeline coniferous species based on xylogenesis observations  yielded mean date of cambial reactivation at the end of April or beginning of May, which agrees with local observations  and very high soil water drainage–are also realistic for treeline with low canopy cover and steep slopes. Although the climate of the study region is cold and rainy, the growth of trees is not limited by high soil moisture content (3 = M4 = 1).The set of estimated VS-model parameters is realistic in terms of physiological thresholds of ironment . The estrvations . Regardi content . This isThe only simulated phenological phases with significant trends are the dates of cambial cell growth onset (0.28 d/y) and the first occurrence of a differentiating cell (0.34 d/y). Those values are noteworthy higher than estimates based purely on climatic data from our study region . Our study site–similarly to the majority of treelines in the Central Europe (1 < T < T2). By contrast, temperature increase in summer results in more frequent occurrence of climatically optimal days with growth rates decoupled from the course of temperature (T2 < T < T3). This coupling/decoupling of spring/summer growth rates from the climatic conditions might have a significant consequences for the temporal stability of the climatic signal stored in tree-ring width chronologies  and intra-annual cambial dynamics . By contrast, the simulated date of the last enlarging cell appearance is biased and together with the simulated growing season duration should be interpreted with caution. Challenging future task is to improve the VS-model performance in the late part of growing season while keeping this model widely applicable without the need for rarely available physiological input data.Using the process-based climate-driven VS-model, we showed that the pronounced growth increase recently observed in treeline stands of Central Europe results mainly from increased growth rates during early and peak growing season. Summer growth rates follow increasing trends of temperatures and become substantially less temperature-limited. In addition, earlier onset of cambial activity and higher spring temperatures promote increasing spring growth rates, which, however, remain mostly temperature-limited. Given the marginal importance of moisture availability as growth-limiting factor, the further increase of intra-annual growth rates and annual tree-ring widths might be anticipated at treeline with ongoing climate change.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JT developed the idea of the study, analyzed the data , and led writing of the manuscript. VT, HK, and JK performed the fieldwork. All authors contributed to writing of the manuscript and approved its final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.