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Backyard production systems (BPS) are distributed worldwide, rearing animals recognized as reservoirs of Salmonella enterica and Shiga toxin-producing Escherichia coli (STEC), both zoonotic pathogens. The aim of this study was to characterize isolates of both pathogens obtained from animals raised in BPS from two central Chile regions. The presence of pathogens was determined by bacterial culture and confirmatory PCR for each sampled BPS, calculating positivity rates. Multivariate logistic regression was used to determine risk factors. Additionally, phenotypic antimicrobial resistance was determined. A positivity rate of 2.88% for S. enterica and 14.39% for STEC was determined for the complete study region (Valparaíso and Metropolitana regions). Risk factor analysis suggests that the presence of ruminants (OR = 1.03; 95% CI = 1.002-1.075) increases the risk of STEC-positive BPS, and the presence of ruminants (OR = 1.05; 95% CI = 1.002-1.075) and the animal handlers being exclusively women (OR = 3.54; 95% CI = 1.029-12.193) increase the risk for S. enterica /STEC positivity. Eighty percent of S. enterica isolates were multidrug resistant, and all STEC were resistant to Cephalexin. This study evidences the circulation of multidrug-resistant zoonotic bacterial strains in animals kept in BPS and the presence of factors that modify the risk of BPS positivity for both pathogens.

Environmental biophysical interactions are recognized to play an essential part in the human biological processes associated with trauma recovery. Many studies over several decades have furthered our understanding of the effects that Pulsed Electromagnetic Fields (PEMF) have on the human body, as well as on cellular and biophysical systems. These investigations have been driven by the observed positive clinical effects of this non-invasive treatment on patients, mainly in orthopedics. Unfortunately, the diversity of the various study setups, with regard to physical parameters, molecular and cellular response, and clinical outcomes, has made it difficult to interpret and evaluate commonalities, which could, in turn, lead to finding an underlying mechanistic understanding of this treatment modality. In this review, we give a birds-eye view of the vast landscape of studies that have been published on PEMF, presenting the reader with a scaffolded summary of relevant literature starting from categorical literature reviews down to individual studies for future research studies and clinical use. We also highlight discrepancies within the many diverse study setups to find common reporting parameters that can lead to a better universal understanding of PEMF effects.

It is widely established that musicians possess a higher level in certain auditory perceptual abilities when compared to non-musicians. This improvement may be mediated, at least in part, by changes in the cochlear response induced by reflex activation of the olivocochlear efferent system. In this review, we describe and analyze the scientific evidence regarding possible differences in the efferent response in musicians and non-musicians. The main evidence observed is that musicians present a greater robustness of the efferent olivocochlear reflex when measured by suppression of otoacoustic emissions and compared to non-musicians. Analyzing the articles presented in this review, it is possible to point out that the differential role of the efferent effect in musicians is not yet established. There is not enough evidence to support the idea that the olivocochlear system favors comparative changes in the properties of musicians' auditory filters. New studies with psychoacoustic techniques, among others, are needed to measure the effect of the olivocochlear reflex on tuning, gain, compression, or temporal resolution in musicians and non-musicians.

To understand the importance of terrestrial solar exposure on human skin, not only individual spectral components need to be considered in biomedical studies, but also the relevance of the combined action profile of the complete solar spectrum (cSS) must be established. We therefore developed a novel irradiation device that combines the emission of four individual lamps (UVB, UVA, VIS and nIR) to achieve exposure from 280 to 1400 nm with individual controllable lamps. The integrated irradiance of each spectral band is similar to the solar spectrum. The lamps can be utilised individually or in any desired combination. Here we present the design, realisation, and validation of this irradiation device as well as biological results on cellular metabolism (MTT assay), cell cycle alterations, and clonogenic growth in HaCaT cells after exposures to the individual spectral bands as well as their simultaneous combinations. Thereby, we demonstrate that UVB combined with UVA is the main determinant for the metabolic activity within cSS. Also, UVB-dependent effects dominate cell cycle regulation in cSS, whilst UVA and nIR have little influence. Lastly, also clonogenic growth is dominated by the UVB action profile in cSS, despite nIR showing modulatory activity when applied in combination with UVB. Together, this highlights the regulatory influence of the different spectral bands on the three biological endpoints and demonstrates their modulation when being part of the complete solar spectrum., (© 2022. The Author(s).)

The BRAF inhibitor vemurafenib, approved for treating patients with BRAF V600E-mutant and unresectable or metastatic melanomas, rapidly induces cutaneous adverse events, including hyperkeratotic skin lesions and cutaneous squamous cell carcinomas (cSCC). To determine, how vemurafenib would provoke these adverse events, we utilized long-term in vitro skin equivalents (SEs) comprising epidermal keratinocytes and dermal fibroblasts in their physiological environment. We inserted keratinocytes with different genetic background [normal keratinocytes: NHEK, HaCaT (p53/mut), and HrasA5 (p53/mut+Hras/mut)] to analyze effects depending on the stage of carcinogenesis. We now show that vemurafenib activates MEK-ERK signaling in both, keratinocytes, and fibroblasts in vitro and in the in vivo -like SEs. As a consequence, vemurafenib does not provide a growth advantage but leads to a differentiation phenotype, causing accelerated differentiation and hyperkeratosis in the NHEK and normalized stratification and cornification in the transformed keratinocytes. Although all keratinocytes responded very similarly to vemurafenib in their expression profile, particularly with a significant induction of MMP1 and MMP3, only the HrasA5 cells revealed a vemurafenib-dependent pathophysiological shift to tumor progression, i.e., the initiation of invasive growth. This was shown by increased proteolytic activity allowing for penetration of the basement membrane and invasion into the disrupted underlying matrix. Blocking MMP activity, by the addition of ilomastat, prevented invasion with all corresponding degradative activities, thus substantiating that the RAS-RAF-MEK-ERK/MMP axis is the most important molecular basis for the rapid switch towards tumorigenic conversion of the HrasA5 keratinocytes upon vemurafenib treatment. Finally, cotreatment with vemurafenib and the MEK inhibitor cobimetinib prevented MEK-ERK hyperactivation and with that abolished both, the epidermal differentiation and the tumor invasion phenotype. This suggests that both cutaneous adverse events are under direct control of vemurafenib-dependent MEK-ERK hyperactivation and confirms the dependence on preexisting genetic alterations of the skin keratinocytes that determine the basis towards induction of tumorigenic progression., Competing Interests: 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., (Copyright © 2022 Tham, Stark, Jauch, Harwood, Pavez Lorie and Boukamp.)

The aim of personalized medicine is to detach from a "one-size fits all approach" and improve patient health by individualization to achieve the best outcomes in disease prevention, diagnosis and treatment. Technological advances in sequencing, improved knowledge of omics, integration with bioinformatics and new in vitro testing formats, have enabled personalized medicine to become a reality. Individual variation in response to environmental factors can affect susceptibility to disease and response to treatments. Space travel exposes humans to environmental stressors that lead to physiological adaptations, from altered cell behavior to abnormal tissue responses, including immune system impairment. In the context of human space flight research, human health studies have shown a significant inter-individual variability in response to space analogue conditions. A substantial degree of variability has been noticed in response to medications (from both an efficacy and toxicity perspective) as well as in susceptibility to damage from radiation exposure and in physiological changes such as loss of bone mineral density and muscle mass in response to deconditioning. At present, personalized medicine for astronauts is limited. With the advent of longer duration missions beyond low Earth orbit, it is imperative that space agencies adopt a personalized strategy for each astronaut, starting from pre-emptive personalized pre-clinical approaches through to individualized countermeasures to minimize harmful physiological changes and find targeted treatment for disease. Advances in space medicine can also be translated to terrestrial applications, and vice versa. This review places the astronaut at the center of personalized medicine, will appraise existing evidence and future preclinical tools as well as clinical, ethical and legal considerations for future space travel., Competing Interests: The handling editor declared a past collaboration with one of the authors MM. The remaining 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., (Copyright © 2021 Pavez Loriè, Baatout, Choukér, Buchheim, Baselet, Dello Russo, Wotring, Monici, Morbidelli, Gagliardi, Stingl, Surdo and Yip.)

In the Metropolitana region of Chile there are 3836 backyard production systems (BPS), characterized as small-scale systems. They act as a source of zoonotic pathogens, such as Salmonella enterica and Shiga toxin-producing Escherichia coli (STEC), whose prevalence in BPS has not been fully described. The objective of this study was to determine the positivity for both agents in BPS and to establish the risk factors related to their presence. In each BPS, an epidemiological survey was undertaken, and stool samples were collected to detect these pathogens via bacteriological culture and conventional PCR techniques. Subsequently, multivariable logistic regression models were applied to establish the risk factors associated with their presence. BPS positivity rates of 11.76% for STEC and 4.7% for S. enterica were observed. The systems showed poor welfare standards and a lack of biosecurity measures. The risk factor analysis concluded that the Gini-Simpson index ( p = 0.030; OR = 1.717) and the presence of neighboring intensive poultry or swine production systems ( p = 0.019; OR = 20.645) act as factors that increased the risk of positivity with respect to STEC. In the case of S. enterica , exchanging embryonated eggs ( p = 0.021; OR = 39) and the presence of debeaked chickens ( p = 0.001; OR = 156) were determined as factors that increased the risk of positivity for this agent. For positivity with respect to both pathogens, the Gini-Simpson index ( p = 0.030; OR = 1.544) and being INDAP/PRODESAL users ( p = 0.023; OR = 15.026) were determined as factors that increased the risk, whereas the type of confinement ( p = 0.002; OR = 0.019) decreased it. Epidemiological surveillance of these neglected populations is lacking, highlighting the fact that STEC and S. enterica maintenance on BPS represents a potential threat to public health.

Porous multiwell plate inserts are widely used in biomedical research to study transport processes or to culture cells/tissues at the air-liquid interface. These inserts are made of rigid materials and used under static culture conditions, which are unrepresentative of biological microenvironments. Here, we present FleXert, a soft, actuatable cell culture insert that interfaces with six-well plates. It is made of polydimethylsiloxane (PDMS) and comprises a porous PDMS membrane as cell/tissue support. FleXerts can be pneumatically actuated using a standard syringe pump, imparting tensile strains of up to 30%. A wide range of actuation patterns can be achieved by varying the air pressure and pumping rate. Facile surface functionalization of FleXert's porous PDMS membrane with fibronectin enables adhesion of human dermal fibroblasts and strains developing on FleXert's membrane are successfully transduced to the cell layer. 3D tissue models, such as fibroblast-laden collagen gels, can also be anchored to PDMS following polydopamine coating. Furthermore, collagen-coated FleXert membranes support the establishment of a human skin model, demonstrating the material's excellent biocompatibility required for tissue engineering. In contrast to existing technologies, FleXerts do not require costly fabrication equipment or custom-built culture chambers, making them a versatile and low-cost solution for tissue engineering and biological barrier penetration studies under physiological strain. This paper is an extensive toolkit for multidisciplinary mechanobiology studies, including detailed instructions for a wide variety of methods such as device fabrication, theoretical modeling, cell culture, and image analysis techniques.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen and important cause of foodborne disease worldwide. Many animal species in backyard production systems (BPS) harbor STEC, systems characterized by low biosecurity and technification. No information is reported on STEC circulation, antimicrobial resistance (AMR) and potential drivers of antimicrobial usage in Chilean BPS, increasing the risk of maintenance and transmission of zoonotic pathogens and AMR generation. Thus, the aim of this study was to characterize phenotypic and genotypic AMR and to study the epidemiology of STEC isolated in BPS from Metropolitana region, Chile. A total of 85 BPS were sampled. Minimal inhibitory concentration and whole genome sequencing was assessed in 10 STEC strain isolated from BPS. All strains were cephalexin-resistant (100%, n = 10), and five strains were resistant to chloramphenicol (50%). The most frequent serotype was O113:H21 (40%), followed by O76:H19 (40%), O91:H14 (10%), and O130:H11 (10%). The stx 1 type was detected in all isolated strains, while stx 2 was only detected in two strains. The Stx subtype most frequently detected was stx 1c (80%), followed by stx 1a (20%), stx 2b (10%), and stx 2d (10%). All strains harbored chromosomal bla AmpC . Principal component analysis shows that BPS size, number of cattle, pet and horse, and elevation act as driver of antimicrobial usage. Logistic multivariable regression shows that recognition of diseases in animals ( p = 0.038; OR = 9.382; 95% CI: 1.138-77.345), neighboring poultry and/or swine BPS ( p = 0.006; OR = 10.564; 95% CI: 1.996-55.894), visit of Veterinary Officials ( p = 0.010; OR = 76.178; 95% CI: 2.860-2029.315) and close contact between animal species in the BPS ( p = 0.021; OR = 9.030; 95% CI: 1.385-58.888) increase significantly the risk of antimicrobial use in BPS. This is the first evidence of STEC strains circulating in BPS in Chile, exhibiting phenotypic AMR, representing a threat for animal and public health. Additionally, we identified factors acting as drivers for antimicrobial usage in BPS, highlighting the importance of integration of these populations into surveillance and education programs to tackle the potential development of antimicrobial resistance and therefore the risk for ecosystemic health., Competing Interests: 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., (Copyright © 2021 Pavez-Muñoz, González, Fernández-Sanhueza, Sánchez, Escobar, Ramos, Fuenzalida, Galarce, Arriagada, Neira, Muñoz-Aguayo, Flores-Figueroa, Johnson and Alegría-Morán.)

We here present the spontaneously immortalised cell line, HaSKpw, as a novel model for the multistep process of skin carcinogenesis. HaSKpw cells were established from the epidermis of normal human adult skin that, without crisis, are now growing unrestricted and feeder-independent. At passage 22, clonal populations were established and clone7 (HaSKpwC7) was further compared to the also spontaneously immortalized HaCaT cells. As important differences, the HaSKpw cells express wild-type p53, remain pseudodiploid, and show a unique chromosomal profile with numerous complex aberrations involving chromosome 20. In addition, HaSKpw cells overexpress a pattern of genes and miRNAs such as KRT34, LOX, S100A9, miR21, and miR155; all pointing to a tumorigenic status. In concordance, HaSKpw cells exhibit reduced desmosomal contacts that provide them with increased motility and a highly migratory/invasive phenotype as demonstrated in scratch- and Boyden chamber assays. In 3D organotypic cultures, both HaCaT and HaSKpw cells form disorganized epithelia but only the HaSKpw cells show tumorcell-like invasive growth. Together, HaSKpwC7 and HaCaT cells represent two spontaneous (non-genetically engineered) "premalignant" keratinocyte lines from adult human skin that display different stages of the multistep process of skin carcinogenesis and thus represent unique models for analysing skin cancer development and progression.

Three-dimensional (3D) in vitro skin and skin cancer models have become an invaluable tool in skin research. They go back to 1979, when Bell and colleagues reported on the establishment of a fibroblast-dependent collagen tissue (Bell, Ivarsson, & Merrill, 1979). On top of such tissue a stratified and differentiated epidermis could be established (Bell, Merrill, & Solomon, 1979). Hydrogel-based dermal equivalents have been generated ever since and upon co-culture with normal human skin keratinocytes, these constructs were then termed skin equivalents. Due to a number of deficiencies, the most important one being their restricted survival time, new developments helped to circumvent premature fibroblast activation and tissue destruction. By avoiding collagen for the dermal equivalent (DE), we proposed, a scaffold-based DE, allowing fibroblasts to reorganize the primary fibrin solution into an "authentic" dermal matrix (Boehnke et al., 2007; Stark et al., 2004, 2006). With this, our goal of a long-term skin equivalent-successful cultivation for several months-was achieved. Nevertheless, also this model presented limitations. One being its opaqueness made it difficult to image the intact tissue. Another draw-back was that tumor cells upon invasion used the scaffold as a guardrail leaving behind an unspecific invasion pattern. All this could be avoided by an approach, the fibroblast-derived matrix-based model, based on the work by Ahlfors and Billiar (2007) We here provide a protocol for this type of model, thereby providing the basis for future work in the field of skin research., (© 2020 Elsevier Inc. All rights reserved.)

There are two types of sensory cells in the mammalian cochlea, inner hair cells, which make synaptic contact with auditory-nerve afferent fibers, and outer hair cells that are innervated by crossed and uncrossed medial olivocochlear (MOC) efferent fibers. Contralateral acoustic stimulation activates the uncrossed efferent MOC fibers reducing cochlear neural responses, thus modifying the input to the central auditory system. The chinchilla, among all studied mammals, displays the lowest percentage of uncrossed MOC fibers raising questions about the strength and frequency distribution of the contralateral-sound effect in this species. On the other hand, MOC effects on cochlear sensitivity have been mainly studied in anesthetized animals and since the MOC-neuron activity depends on the level of anesthesia, it is important to assess the influence of anesthesia in the strength of efferent effects. Seven adult chinchillas (Chinchilla laniger) were chronically implanted with round-window electrodes in both cochleae. We compared the effect of contralateral sound in awake and anesthetized condition. Compound action potentials (CAP) and cochlear microphonics (CM) were measured in the ipsilateral cochlea in response to tones in absence and presence of contralateral sound. Control measurements performed after middle-ear muscles section in one animal discarded any possible middle-ear reflex activation. Contralateral sound produced CAP amplitude reductions in all chinchillas, with suppression effects greater by about 1-3 dB in awake than in anesthetized animals. In contrast, CM amplitude increases of up to 1.9 dB were found in only three awake chinchillas. In both conditions the strongest efferent effects were produced by contralateral tones at frequencies equal or close to those of ipsilateral tones. Contralateral CAP suppressions for 1-6 kHz ipsilateral tones corresponded to a span of uncrossed MOC fiber innervation reaching at least the central third of the chinchilla cochlea.

Terminal differentiation of keratinocytes is a multistep process that requires a coordinated program of gene expression. We aimed to explore the possible involvement of a previously unreported class of non-coding RNA genes, microRNAs (miRNAs) in keratinocyte differentiation by using miRNA expression profiling. Out of 365 miRNAs tested, 7 showed significant change between keratinocytes cultured in low or high calcium concentration. The highest-ranked upregulated gene was miR-203, whose expression was significantly upregulated in response to calcium and other inducers of keratinocyte differentiation such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and vitamin D(3). Differentiation-induced upregulation of miR-203 expression was blocked by treatment with specific inhibitors of protein kinase C (PKC), GF109203X, and Ro31-8220. Moreover, our results showed that the activator protein-1 (AP-1) proteins c-Jun and JunB regulate miR-203 expression in keratinocytes. In contrast to inducers of keratinocyte differentiation, epidermal growth factor and keratinocyte growth factor suppressed miR-203 expression in keratinocytes below the basal level. Overexpression of miR-203 in keratinocytes resulted in enhanced differentiation, whereas inhibition of miR-203 suppressed calcium-induced terminal differentiation as judged by involucrin expression. These results suggest that upregulation of miR-203 in human keratinocytes is required for their differentiation and is dependent on the activation of the PKC/AP-1 pathway.

The biosynthesis of retinoic acid (RA) from retinol is controlled by several enzymes, e.g. dehydrogenases (RalDH2, RoDH-4) and retinol-esterifying enzyme (LRAT), whereas its degradation mainly involves CYP26 enzymes. In keratinocytes, RA activates the nuclear retinoid-receptors inducing the transcription of many genes. Here, we examined the effects of RA and the CYP26 inhibitors, liarozole and talarozole, on retinoid metabolism and RA-regulated genes in organotypic epidermis. RA induced the expression of CYP26 enzymes already after 8 h, whereas LRAT exhibited a later response and peaked at 48 h, indicating a feedback induction of retinol esterification. In line with a reduced biosynthesis of RA from retinol after exogenous RA, the expression of RDH16 reduced 80% in response to exogenous RA. The mRNA expression of RA-regulated genes (KRT2, KRT4, CRABPII and HBEGF) was altered within 24 h after RA exposure. In contrast, the CYP26 inhibitors caused only minor effects, except for a clear-cut induction of CYP26A1 only when combined with minute amounts of exogenous RA. Cellular accumulation of exogenous [3H]RA was higher after talarozole than after liarozole, probably indicating a greater CYP26-inhibitory potency of the former drug. The present study shows that CYP26A1 expression is extremely sensitive to both exogenous RA and increased endogenous RA levels, i.e. due to CYP26 inhibition, and thus an excellent biomarker for retinoid signalling in organotypic epidermis.

All-trans retinoic acid (RA) levels are controlled by enzymes of the vitamin A metabolism (RDH16, RalDH2, and LRAT) and RA catabolism (CYP26 and CYP2S1). Here, the mRNA expression of these enzymes was investigated in human keratinocytes at different Ca(2+)concentrations and after exposure to RA and CYP26 inhibitors. Cellular differentiation (high Ca(2+)) increased the expression of LRAT, RDH16 and RalDH2, and decreased CYP26B1. RA (1 microM) induced CYP26A1, CYP26B1, CYP2S1, CRABPII and LRAT mRNA. The CYP26 inhibitor talarozole altered CYP26A1 and LRAT mRNA expression in a similar way as RA, increased the cellular accumulation of [(3)H]RA, and induced a punctate CRABPII staining, also observed after siRNA knock-down of CYP26B1 (but not after RA exposure). Furthermore, CYP26B1 siRNA increased the accumulation of [(3)H]RA and the CRABPII mRNA, suggesting an augmented retinoid signalling. Thus CYP26B1 appears essential for RA catabolism under physiological conditions, whereas CYP26A1 might play a greater role during RA excess.

Lamellar ichthyosis is a keratinization disorder caused by TGM1, Ichthyin and several other gene mutations. A new treatment option is liarozole, which blocks the cytochrome P450 (CYP26)-mediated catabolism of endogenous all-trans retinoic acid. This study focuses on the expression of retinoid-related genes in ichthyotic epidermis before and after treatment with oral liarozole. We first compared the mRNA expression of cellular retinoic acid binding protein II (CRABPII), keratin (KRT) 2 and 4, CYP26A1 and B1, and two markers of inflammation (interleukin-1alpha and tumours necrosis factor (TNF)-alpha) in shave biopsies from 11 genetically defined, untreated patients and 12 age- and sex-matched healthy controls, finding no overt differences between the groups, besides elevated CRABPII expression. We then studied the biomarkers before and after 4 weeks of treatment with liarozole (75 or 150 mg/day), which produced a better therapeutic response in patients with Ichthyin (n=3) than in those with TGM1 (n=6) mutations. A significant decrease in the mRNA expression of KRT2 and TNF-alpha, and trends toward increased expression of KRT4 and CYP26A1 were observed in liarozole-treated patients, consistent with an increased retinoid stimulation of epidermis. However, there were no dose-related responses and the results of the immunostaining did not always parallel the mRNA findings. The results suggest that liarozole exerts a therapeutic effect in lamellar ichthyosis by mildly affecting the expression of retinoid- regulated genes in epidermis.

Besides the intensity and frequency of an auditory stimulus, the length of time that precedes the stimulation is an important factor that determines the magnitude of early evoked neural responses in the auditory cortex. Here we used chinchillas to demonstrate that the length of the silent period before the presentation of an auditory stimulus is a critical factor that modifies late oscillatory responses in the auditory cortex. We used tetrodes to record local-field potential (LFP) signals from the left auditory cortex of ten animals while they were stimulated with clicks, tones or noise bursts delivered at different rates and intensity levels. We found that the incidence of oscillatory activity in the auditory cortex of anesthetized chinchillas is dependent on the period of silence before stimulation and on the intensity of the auditory stimulus. In 62.5% of the recordings sites we found stimulus-related oscillations at around 8-20 Hz. Stimulus-induced oscillations were largest and consistent when stimuli were preceded by 5 s of silence and they were absent when preceded by less than 500 ms of silence. These results demonstrate that the period of silence preceding the stimulus presentation and the stimulus intensity are critical factors for the presence of these oscillations.