Cellular calcium handling and electrophysiology are modulated by chronic physiological pacing in human induced pluripotent stem cell-derived cardiomyocytes.

Electric pacing of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) has been increasingly used to simulate cardiac arrhythmias in vitro and to enhance cardiomyocyte maturity. However, the impact of electric pacing on cellular electrophysiology and Ca2+-handling in differentiated hiPSC-CM is less characterized. Here we studied the effects of electric pacing for 24h or 7d at a physiological rate of 60 bpm on cellular electrophysiology and Ca2+-cycling in late-stage, differentiated hiPSC-CM (>90% troponin+, >60d post differentiation). Electric culture pacing for 7d did not influence cardiomyocyte cell size, apoptosis or generation of reactive oxygen species in differentiated hiPSC-CM compared to 24h pacing. However, epifluorescence measurements revealed that electric pacing for 7d improved systolic Ca2+-transient amplitude and Ca2+-transient upstroke, which could be explained by elevated sarcoplasmic reticulum Ca2+-load and SERCA activity. Diastolic Ca2+-leak was not changed in line-scanning confocal microscopy suggesting that the improvement in systolic Ca2+-release was not associated with a higher open probability of RyR2 during diastole. While bulk cytosolic Na+-concentration and NCX activity were not changed, patch-clamp studies revealed that chronic pacing caused a slight abbreviation of the action potential duration (APD) in hiPSC-CM. We found in whole-cell voltage-clamp measurements that chronic pacing for 7d led to a decrease in late Na+-current, which might explain the changes in APD. In conclusion, our results show that chronic pacing improves systolic Ca2+-handling and modulates the electrophysiology of late-stage, differentiated iPSC-CM. This study might help to understand the effects of electric pacing and its numerous applications in stem cell research including arrhythmia simulation.

Survival and Neurologic Outcomes From Pharmacologic Peptide Administration During Cardiopulmonary Resuscitation of Pulseless Electrical Activity.

BACKGROUND: Outcomes from cardiopulmonary resuscitation (CPR) following sudden cardiac arrest are suboptimal. Postresuscitation targeted temperature management has been shown to have benefit in subjects with sudden cardiac arrest due to ventricular fibrillation, but there are few data for outcomes from sudden cardiac arrest due to pulseless electrical activity. In addition, intra-CPR cooling is more effective than postresuscitation cooling. Physical cooling is associated with increased protein kinase B activity. Therefore, our group developed a novel peptide, TAT-PHLPP9c, which regulates protein kinase B. We hypothesized that when given during CPR, TAT-PHLPP9c would improve survival and neurologic outcomes following pulseless electrical activity arrest. METHODS AND RESULTS: In 24 female pigs, pulseless electrical activity was induced by inflating balloon catheters in the right coronary and left anterior descending arteries for ≈7 minutes. Advanced life support was initiated. In 12 control animals, epinephrine was given after 1 and 3 minutes. In 12 peptide-treated animals, 7.5 mg/kg TAT-PHLPP9c was also administered at 1 and 3 minutes of CPR. The balloons were removed after 2 minutes of support. Animals were recovered and neurologically scored 24 hours after return of spontaneous circulation. Return of spontaneous circulation was more common in the peptide group, but this difference was not significant (8/12 control versus 12/12 peptide; P=0.093), while fully intact neurologic survival was significantly more common in the peptide group (0/12 control versus 11/12 peptide; P<0.00001). TAT-PHLPP9c significantly increased myocardial nicotinamide adenine dinucleotide levels. CONCLUSIONS: TAT-PHLPP9c resulted in improved survival with full neurologic function after sudden cardiac arrest in a swine model of pulseless electrical activity, and the peptide shows potential as an intra-CPR pharmacologic agent.

Synthetic Approaches to the New Drugs Approved During 2022.

In 2022, 23 new small molecule chemical entities were approved as drugs by the United States FDA, European Union EMA, Japan PMDA, and China NMPA. This review describes the synthetic approach demonstrated on largest scale for each new drug based on patent or primary literature. The synthetic routes highlight practical methods to construct molecules, sometimes on the manufacturing scale, to access the new drugs. Ten additional drugs approved in 2021 and one approved in 2020 are included that were not covered in the previous year's review.

A standardized wound infection model for antimicrobial testing of wound dressings in vitro.

To investigate the effectiveness of antimicrobial agents against wound infections, experiments using either 2D cultures with planktonic microorganisms or animal infection models are frequently carried out. However, the transferability of the results to human skin is limited by the lack of complexity of the 2D models or by the poor translation of the results from animal models. Hence, there is a need for wound infection models capable of assessing antimicrobial agents. In this study, an easily standardized wound infection model was established. This model consists of a mechanically wounded human skin model on a collagen matrix infected with various clinically relevant bacteria. Infection of the model led to recognition of the pathogens and induction of an inflammatory response. The untreated infection spread over time, causing significant tissue damage. By applying an antimicrobial-releasing wound dressing, the bacterial load could be reduced and the success of the treatment could be further measured by a decrease in the inflammatory reaction. In conclusion, this wound infection model can be used to evaluate new antimicrobial therapeutics as well as to study host-pathogen interactions.

Synthetic Approaches to the New Drugs Approved During 2021.

Each year, new drugs are introduced to the market, representing structures that have affinity for biological targets implicated in human diseases and conditions. These new chemical entities (NCEs), particularly small molecules and antibody-drug conjugates, provide insight into molecular recognition and serve as potential leads for the design of future medicines. This annual review is part of a continuing series highlighting the most likely process-scale synthetic approaches to 35 NCEs that were first approved anywhere in the world during 2021.

Cold Atmospheric Plasma Exerts Antimicrobial Effects in a 3D Skin Model of Cutaneous Candidiasis.

Cutaneous candidiasis is characterized by an overgrowth of Candida leading to skin inflammation and infection. Similar to bacteria, Candida can develop tolerance to common antifungal drugs. Cold atmospheric plasma (CAP), with its proven antimicrobial properties, offers a promising alternative to the prevailing methods. Because of plasma heterogeneity each new device must be tested individually for its effectiveness. Antimicrobial activity is usually studied using planktonic microorganisms or animal models, making it difficult to extrapolate the results to the human system. Therefore, a 3D skin model of cutaneous candidiasis for the antimicrobial testing of CAP was established. First, the reaction of the 3D-skin model to Candida infection was examined using various histological and molecular-biological methods. Infection with C. albicans resulted in increased expression and secretion of pro-inflammatory cytokines and augmented expression of antimicrobial peptides. Within 48 h, hyphal growth spread throughout the model and caused tissue damage. Second, the CAP treatment was employed. It was shown that CAP significantly reduced the spread of the yeast in the infected skin models as well as decreased the expression and secretion of the infection markers. The plasma device exhibited a high antifungal activity by completely inhibiting hyphal growth and reducing inflammation at the highest treatment duration.

A cell-penetrating PHLPP peptide improves cardiac arrest survival in murine and swine models.

Out-of-hospital cardiac arrest is a leading cause of death in the US, with a mortality rate over 90%. Preclinical studies demonstrate that cooling during cardiopulmonary resuscitation (CPR) is highly beneficial, but can be challenging to implement clinically. No medications exist for improving long-term cardiac arrest survival. We have developed a 20-amino acid peptide, TAT-PHLPP9c, that mimics cooling protection by enhancing AKT activation via PH domain leucine-rich repeat phosphatase 1 (PHLPP1) inhibition. Complementary studies were conducted in mouse and swine. C57BL/6 mice were randomized into blinded saline control and peptide-treatment groups. Following a 12-minute asystolic arrest, TAT-PHLPP9c was administered intravenously during CPR and significantly improved the return of spontaneous circulation, mean arterial blood pressure and cerebral blood flow, cardiac and neurological function, and survival (4 hour and 5 day). It inhibited PHLPP-NHERF1 binding, enhanced AKT but not PKC phosphorylation, decreased pyruvate dehydrogenase phosphorylation and sorbitol production, and increased ATP generation in heart and brain. TAT-PHLPP9c treatment also reduced plasma taurine and glutamate concentrations after resuscitation. The protective benefit of TAT-PHLPP9c was validated in a swine cardiac arrest model of ventricular fibrillation. In conclusion, TAT-PHLPP9c may improve neurologically intact cardiac arrest survival without the need for physical cooling.

Synthetic Approaches to the New Drugs Approved During 2020.

New drugs introduced to the market are privileged structures that have affinities for biological targets implicated in human diseases and conditions. These new chemical entities (NCEs), particularly small molecules and antibody-drug conjugates (ADCs), provide insight into molecular recognition and simultaneously function as leads for the design of future medicines. This Review is part of a continuing series presenting the most likely process-scale synthetic approaches to 44 new chemical entities approved for the first time anywhere in the world during 2020.

Aurora B activity is promoted by cooperation between discrete localization sites in budding yeast.

Chromosome biorientation is promoted by the four-member chromosomal passenger complex (CPC) through phosphorylation of incorrect kinetochore-microtubule attachments. During chromosome alignment, the CPC localizes to the inner centromere, the inner kinetochore, and spindle microtubules. Here we show that a small domain of the CPC subunit INCENP/Sli15 is required to target the complex to all three of these locations in budding yeast. This domain, the single alpha helix (SAH), is essential for phosphorylation of outer kinetochore substrates, chromosome segregation, and viability. By restoring the CPC to each of its three locations through targeted mutations and fusion constructs, we determined their individual contributions to chromosome biorientation. We find that only the inner centromere localization is sufficient for cell viability on its own. However, when combined, the inner kinetochore and microtubule binding activities are also sufficient to promote accurate chromosome segregation. Furthermore, we find that the two pathways target the CPC to different kinetochore attachment states, as the inner centromere-targeting pathway is primarily responsible for bringing the complex to unattached kinetochores. We have therefore discovered that two parallel localization pathways are each sufficient to promote CPC activity in chromosome biorientation, both depending on the SAH domain of INCENP/Sli15.

Establishing a multicenter, preclinical consortium in resuscitation: A pilot experimental trial evaluating epinephrine in cardiac arrest.

BACKGROUND: Large animal studies are an important step in the translation pathway, but single laboratory experiments do not replicate the variability in patient populations. Our objective was to demonstrate the feasibility of performing a multicenter, preclinical, randomized, double-blinded, placebo-controlled cardiac arrest trial. We evaluated the effect of epinephrine on coronary perfusion pressure (CPP) as previous single laboratory studies have reported mixed results. METHODS: Forty-five swine from 5 different laboratories (Ann Arbor, MI; Baltimore, MD; Los Angeles, CA; Pittsburgh, PA; Toronto, ON) using a standard treatment protocol. Ventricular fibrillation was induced and left untreated for 6 min before starting continuous cardiopulmonary resuscitation (CPR). After 2 min of CPR, 9 animals from each lab were randomized to 1 of 3 interventions given over 12 minutes: (1) Continuous IV epinephrine infusion (0.00375 mg/kg/min) with placebo IV normal saline (NS) boluses every 4 min, (2) Continuous placebo IV NS infusion with IV epinephrine boluses (0.015 mg/kg) every 4 min or (3) Placebo IV NS for both infusion and boluses. The primary outcome was mean CPP during the 12 mins of drug therapy. RESULTS: There were no significant differences in mean CPP between the three groups: 14.4 ± 6.8 mmHg (epinephrine Infusion), 16.9 ± 5.9 mmHg (epinephrine bolus), and 14.4 ± 5.5 mmHg (placebo) (p = NS). Sensitivity analysis demonstrated inter-laboratory variability in the magnitude of the treatment effect (p = 0.004). CONCLUSION: This study demonstrated the feasibility of performing a multicenter, preclinical, randomized, double-blinded cardiac arrest trials. Standard dose epinephrine by bolus or continuous infusion did not increase coronary perfusion pressure during CPR when compared to placebo.

In vitro investigation of the principle of action of ammonium bituminosulfonate ointments on a 3D skin model.

Ammonium bituminosulfonate preparations have been used for various dermatological diseases since the 19th century. The dark preparation is known as the so-called "drawing salve" (Ichtholan®) for the treatment of abscesses and furuncles. The underlying activity is in part the loosening of the skin, which facilitates pus extraction and treatment of deep inflammations. For this investigation 3D skin models were incubated with ointments containing different ammonium bituminosulfonate concentrations. Histological and immunohistochemical staining as well as penetration investigation were carried out. The effect of dark ammonium bituminosulfonate ointments on skin loosening was investigated to reveal the underlying mechanism. The skin loosening effect could be proved by HE-staining for ammonium bituminosulfonate treated skin models. This effect was concentration dependent. While treatment with ammonium bituminosulfonate ointment had no influence on keratin expression, high concentrated ointments led to decreased filaggrin and laminin expression. Treatment of skin models with ABS ointments led to an increased skin permeability, which was concentration dependent. For the first time the skin loosening effect of ammonium bituminosulfonate ointment has been demonstrated on 3D skin models. This effect is at least in part caused by the interaction of the substance with structure dependent proteins of the epidermis.

Efficacy of antifungal agents against fungal spores: An in vitro study using microplate laser nephelometry and an artificially infected 3D skin model.

Dermal fungal infections seem to have increased over recent years. There is further a shift from anthropophilic dermatophytes to a growing prevalence of zoophilic species and the emergence of resistant strains. New antifungals are needed to combat these fungi and their resting spores. This study aimed to investigate the sporicidal effects of sertaconazole nitrate using microplate laser nephelometry against the microconidia of Trichophyton, chlamydospores of Epidermophyton, blastospores of Candida, and conidia of the mold Scopulariopsis brevicaulis. The results obtained were compared with those from ciclopirox olamine and terbinafine. The sporicidal activity was further determined using infected three-dimensional full skin models to determine the antifungal effects in the presence of human cells. Sertaconazole nitrate inhibited the growth of dermatophytes, molds, and yeasts. Ciclopirox olamine also had good antifungal activity, although higher concentrations were needed compared to sertaconazole nitrate. Terbinafine was highly effective against most dermatophytes, but higher concentrations were required to kill the resistant strain Trichophyton indotineae. Sertaconazole nitrate, ciclopirox olamine, and terbinafine had no negative effects on full skin models. Sertaconazole nitrate reduced the growth of fungal and yeast spores over 72 h. Ciclopirox olamine and terbinafine also inhibited the growth of dermatophytes and molds but had significantly lower effects on the yeast. Sertaconazole nitrate might have advantages over the commonly used antifungals ciclopirox olamine and terbinafine in combating resting spores, which persist in the tissues, and thus in the therapy of recurring dermatomycoses.

Skin Failure Among Critically Ill Patients Afflicted with Coronavirus Disease 2019 (COVID-19).

Objective: To characterize skin integrity among coronavirus disease 2019 (COVID-19) patients treated in the intensive care unit (ICU), and identify risk factors for skin failure (SF) in these patients. Design: The characteristic, profound pro-inflammatory, hypercoagulable state of COVID-19 is manifested by the high severity of illness and extensive organ dysfunction observed in these patients. SF in critically ill patients, although described previously, exhibits a uniquely complex pathogenesis in this population. Patients: Retrospective review of all COVID-19 patients (confirmed positive for severe acute respiratory syndrome coronavirus-2 [SARS-CoV-2]) admitted to a single surgical ICU for at least 48 hours between March-June 2020. Interventions: Data were extracted from a COVID-19 institutional data repository that harvested data from electronic health records and other clinical data sources. Demographics; coagulation/inflammation biomarkers; number, location, and stage of SF lesions; resource utilization; and outcomes were captured. Measurements and Main Results: 64 patients met inclusion criteria; 51 (80%) developed SF (SF+ ). Forty-three (85%) developed stage 3 or higher SF (χ2 = 22.66, P < .0001). Thirty-nine of 51 (76%) SF+ patients developed more than one SF lesion (χ2 = 13.26, P = .0003). SF+ patients manifested a profound pro-inflammatory, hypercoagulable phenotype (lower serum albumin and higher ferritin, interleukin [IL]-6 and D-dimer concentrations [all, P < .001]). Durations of mechanical ventilation, vasopressor therapy, and ICU length of stay were significantly longer (all, P < .05) in the SF + patients. Conclusions: The unique characteristics of COVID-19 dermatopathology and the strong correlation between markers of inflammation and development of SF reflect COVID-19-related organ dysfunction and its deleterious effects on the microcirculation. Considering that skin is invaded directly by SARS-CoV-2 and affected by COVID-19-related immune complex deposition and microthrombosis, SF may reflect disease as opposed to pressure injuries related to processes of care. In the context of COVID-19 critical illness, SF should not be considered a "never event."

Pulseless Electrical Activity as the Initial Cardiac Arrest Rhythm: Importance of Preexisting Left Ventricular Function.

Background Pulseless electrical activity (PEA) is a common initial rhythm in cardiac arrest. A substantial number of PEA arrests are caused by coronary ischemia in the setting of acute coronary occlusion, but the underlying mechanism is not well understood. We hypothesized that the initial rhythm in patients with acute coronary occlusion is more likely to be PEA than ventricular fibrillation in those with prearrest severe left ventricular dysfunction. Methods and Results We studied the initial cardiac arrest rhythm induced by acute left anterior descending coronary occlusion in swine without and with preexisting severe left ventricular dysfunction induced by prior infarcts in non-left anterior descending coronary territories. Balloon occlusion resulted in ventricular fibrillation in 18 of 34 naïve animals, occurring 23.5±9.0 minutes following occlusion, and PEA in 1 animal. However, all 18 animals with severe prearrest left ventricular dysfunction (ejection fraction 15±5%) developed PEA 1.7±1.1 minutes after occlusion. Conclusions Acute coronary ischemia in the setting of severe left ventricular dysfunction produces PEA because of acute pump failure, which occurs almost immediately after coronary occlusion. After the onset of coronary ischemia, PEA occurred significantly earlier than ventricular fibrillation (<2 minutes versus 20 minutes). These findings support the notion that patients with baseline left ventricular dysfunction and suspected coronary disease who develop PEA should be evaluated for acute coronary occlusion.

Synthetic Approaches to the New Drugs Approved during 2019.

New drugs introduced to the market are privileged structures having affinities for biological targets implicated in human diseases and conditions. These new chemical entities (NCEs), particularly small molecules and antibody-drug conjugates, provide insight into molecular recognition and simultaneously function as leads for the design of future medicines. This review is part of a continuing series presenting the most likely process-scale synthetic approaches to 40 NCEs approved for the first time anywhere in the world in 2019.

A comparison of three conducted electrical weapons in a surrogate swine cardiac safety model.

We used a previously described methodology in a swine model to compare the relative cardiac safety of the Axon T7 Conducted Electrical Weapon (CEW), released in October of 2018, to two prior generations of Axon CEWs to include the X2 and the X26E. A total of 5 swine (252 total CEW exposures) were tested by alternating the three weapons at each chest exposure location. Our testing, using systemic hypotension as the quantitative surrogate for cardiac capture, demonstrated that the T7 and X2 were not statistically different. Both were superior, in terms of reduced hypotension during exposure, to the X26E. This study is important as it demonstrates that the newly released weapon is non-inferior to the X2 and superior to the X26E using this surrogate safety model. It is also important because it is the first study to examine the cardiac effects of simultaneous multi-bay exposures. Our prior study compared the X2 to the X26E but examined only single bay exposures from the X2. Lastly, we feel we have improved the methodology for studying the comparative cardiac effects of CEWs.

Accuracy of custom temporomandibular joint replacement surgery using a virtual surgical planning protocol.

BACKGROUND: Accurate placement of TMJ implant components may be facilitated by virtual surgical planning (VSP) technologies. The aim of this study was to assess the accuracy of a typical VSP protocol and describe the pattern of surgical error associated with total alloplastic TMJ replacement. METHODS: A retrospective analysis was undertaken on 40 adult patients who were implanted with a fully customised, 3D printed TMJ prosthesis due to end-stage TMJ disease. Planned TMJ implant position based on preoperative CBCT images was compared with final position on postoperative OPGs using a previously validated linear rescaling method. Translational discrepancy was described in the anterior-posterior direction and superior-inferior direction. Rotational discrepancy was described as anterior or posterior. RESULTS: Lin's concordance between preoperative and postoperative position was 0.97, with no significant differences (p > 0.05). The Bland-Altman analysis showed a 95% limit of agreement between planned and final position of - 5.9 to 5.4 mm. Overall, final implant position was more anterior (0.4 mm), superior (0.4 mm) and posteriorly rotated (2.4°) compared with planned position. CONCLUSION: The use of VSP in TMJ replacement surgery results in accurate implant placement with good agreement between planned and final implant position. Discrepancies in planned and final implant position tended to result in the mandibular component of the implant being translated anterior superiorly and rotated posteriorly, with potential implications for the biomechanical performance of the implant and overall device longevity. These results should be used to assist TMJ surgeons pre- and intraoperatively to facilitating accurate implant positioning and optimal surgical rehabilitation.

A shell-less hen's egg test as infection model to determine the biocompatibility and antimicrobial efficacy of drugs and drug formulations against Pseudomonas aeruginosa.

A shell-less hen's egg based infection test with Pseudomonas aeruginosa was established to investigate the antimicrobial efficacy of drugs and drug formulations close to the in vivo situation. The test system using preincubated fertilized chicken eggs transferred in petri dishes was optimized with respect to the controlled local application of liquid materials and bacteria as well as the bacterial cultivation conditions. The applicability of the ex ovo infection model was confirmed with antimicrobial susceptibility tests using tobramycin, ciprofloxacin and meropenem. The validity of the ex ovo data was demonstrated by correlation with in vitro data of the CellTiter®-Blue and the microplate laser nephelometry assay. Real-time imaging of the progress of infection and the efficacy of the treatment could be realized by the MolecuLight i:X™ technique. Furthermore, in a proof-of-concept efficacy, biocompatibility and even the presence of irritants were determined side-by-side using commercial ophthalmics. In conclusion, this egg based infection model could bridge the gap between in vitro and in vivo models for the evaluation of antimicrobial susceptibility to reduce animal tests according to the 3R concept.

Accuracy of orthopantomograms in the assessment of implant position following alloplastic temporomandibular joint replacement.

PURPOSE: The aim of this study was to determine the accuracy of orthopantomograms (OPGs) when assessing post-operative temporomandibular joint (TMJ) implant position, compared with cone beam computerized tomography (CBCT). METHODS: A retrospective analysis was undertaken on six adult patients who were implanted with a custom TMJ prosthesis due to end-stage TMJ disease. Post-operative CBCT was compared with post-operative OPGs. Overall magnification of each OPG was calculated and used to linearly rescale each image. Implant position was assessed by measuring the gonion angle and the distance between each surgical screw and the mandibular gonion (SG length). RESULTS: Mean magnification for OPGs was 24.2%. There were no significant differences (p > 0.05) in the gonion angle on OPGs compared with CBCT images. There was a mean decrease in SG lengths of 0.02 mm on OPGs, corresponding to error level of 5.31%. The 95% limits of agreement between OPGs and CBCT images for SG lengths were 1.65 mm and - 1.73 mm. CONCLUSION: This study presents a clinically applicable and accurate first-line radiographic screening tool to assess TMJ implant position. When combined with clinical assessment, OPGs can help reduce the need for further imaging and radiation exposure post-operatively.

Synthetic Approaches to New Drugs Approved during 2018.

New drugs introduced to the market every year represent privileged structures for particular biological targets. These new chemical entities (NCEs) provide insight into molecular recognition while serving as leads for designing future new drugs. This annual review describes the most likely process-scale synthetic approaches to 39 new chemical entities approved for the first time globally in 2018.