A Novel Genetically Encoded Single Use Sensory Cellular Test System Measures Bicarbonate Concentration Changes in Living Cells.

Bicarbonate plays a central role in human physiology from cellular respiration to pH homeostasis. However, so far, the measurement of bicarbonate concentration changes in living cells has only been possible by measuring intracellular pH changes. In this article, we report the development of a genetically encoded pH-independent fluorescence-based single-use sensory cellular test system for monitoring intracellular bicarbonate concentration changes in living cells. We describe the usefulness of the developed biosensor in characterizing the bicarbonate transport activities of anionophores-small molecules capable of facilitating the membrane permeation of this anion. We also demonstrate the ability of the bicarbonate sensory cellular test system to measure intracellular bicarbonate concentration changes in response to activation and specific inhibition of wild-type human CFTR protein when co-expressed with the bicarbonate sensing and reporting units in living cells. A valuable benefit of the bicarbonate sensory cellular test system could be the screening of novel anionophore library compounds for bicarbonate transport activity with efficiencies close to the natural anion channel CFTR, which is not functional in the respiratory epithelia of cystic fibrosis patients.

Up-regulation of two distinct p53-DNA binding functions by covalent poly(ADP-ribosyl)ation: transactivating and single strand break sensing.

We used a [(32)P] p53 sequence-specific oligodeoxynucleotide and Electrophoretic-Mobility-Shift-Assays to monitor p53 DNA sequence-specific binding with p53-R267W, a nonbinding point mutant; and p53-Δ30, a deletion-mutant which lacks the carboxy-terminus that recognizes DNA-strand-breaks. Recombinant p53 and poly(ADP-ribose)polymerase-1 (PARP-1) were incubated with labeled ßNAD(+) with/without DNA. The poly(ADP-ribosyl)ation of each protein increased with incubation-time and ßNAD(+) and p53 concentration(s). Since p53-Δ30 was efficiently labeled, poly(ADP-ribosyl)ation target site(s) of wt-p53 must reside outside its carboxy-terminal-domain. The poly(ADP-ribosyl)ation of p53-Δ30 did not diminish its DNA binding; Instead, it enhanced DNA-sequence-specific-binding. Therefore, we conclude that DNA-sequence-specific-binding and DNA-nick-sensing of mutant-p53 are differentially regulated by poly(ADP-ribosyl)ation.

Prospective study on harvesting autologous bone grafts from the anterior iliac crest using a new specialized reamer.

The iliac crest remains the most frequent donor site for bone harvesting. Despite the surgical access to the iliac crest being relatively simple and the operation being carried out regularly, there are frequent complications. Therefore, a new, manual iliac crest reamer (R group) was compared to the classical harvesting of a corticocancellous bone graft by means of an oscillating saw (Con group) in a prospective study on 80 consecutive patients having hand surgery. Follow-up time was 3 months. Operation time and incidence of hematomas, seromas, and paresthesias in the R group were significantly shorter and less, respectively, than in the Con group. Pain at harvest site measured with the visual analogue scale (VAS) at 5 days, 6 weeks, and 12 weeks postoperatively was significantly less in group R as well. The utilization of the iliac crest reamer allows bone graft harvest in a relatively quick and simple operation with relatively few complications but with the limitation in that the maximum diameter of a bone cylinder that it can harvest is 20 mm.

Application of a Receptor-Binding-Domain-Based Simple Immunoassay for Assessing Humoral Immunity against Emerging SARS-CoV-2 Virus Variants.

We have developed a simple, rapid, high-throughput RBD-based ELISA to assess the humoral immunity against emerging SARS-CoV-2 virus variants. The cDNAs of the His-tagged RBD proteins of the virus variants were stably engineered into HEK cells secreting the protein into the supernatant, and RBD purification was performed by Ni-chromatography and buffer exchange by membrane filtration. The simplified assay uses single dilutions of sera from finger-pricked native blood samples, purified RBD in 96-well plates, and a chromogenic dye for development. The results of this RBD-ELISA were confirmed to correlate with those of a commercial immunoassay measuring antibodies against the Wuhan strain, as well as direct virus neutralization assays assessing the cellular effects of the Wuhan and the Omicron (BA.5) variants. Here, we document the applicability of this ELISA to assess the variant-specific humoral immunity in vaccinated and convalescent patients, as well as to follow the time course of selective vaccination response. This simple and rapid assay, easily modified to detect humoral immunity against emerging SARS-CoV-2 virus variants, may help to assess the level of antiviral protection after vaccination or infection.

Prospective evaluation of a single-sided innervated gluteal artery perforator flap for reconstruction for extensive and recurrent pilonidal sinus disease: functional, aesthetic, and patient-reported long-term outcomes.

BACKGROUND: Treating large and extensive pilonidal sinus disease is a challenging task. Long-term reports on flaps suitable for coverage of large, wide, local-excision defects are sparse. We prospectively evaluated data with a minimum 1-year follow-up of the use of a single-sided, innervated, superior gluteal artery perforator flap. METHODS: Twenty-one patients (1 woman, 20 men) with a median age of 26 years (min - max = 18 - 46) were included in the study period from September 2005 to April 2010. We recorded flap size, major and minor complications, hospital length of stay, impairment in activities of daily living, pain, aesthetic outcomes, and sensibility in the gluteal region (PSSD, pressure-specified sensory device) at 6 and 12 months postoperatively. RESULTS: The mean defect size (±SD) was 13.0 ± 1.9 × 8.6 ± 1.3 × 5.5 ± 1.2 cm (height × width × depth), and median length of hospital stay was 9 days (range = 7-11). Only two patients developed minor wound-healing complications. Visual analog pain scales significantly improved, with no pain detectable at 12 months postoperatively (p < 0.0001). The aesthetic appearance of the results was good in the majority of patients (61.9-85.7 %). PSSD showed gradual normalization, with retained sensibility in the flap area over 12 months postoperatively (p = 0.0232). During the median 36-month (range = 20-60) follow-up, we have not observed any recurrence in the operated area. CONCLUSIONS: The innervated superior gluteal artery perforator flap is a useful technique for covering large and recurrent pilonidal sinus defects following wide local excision and represents an excellent tool in the surgical armamentarium for achieving long-lasting outcomes in this young group of patients.

Evolution of the 3-dimensional video system for facial motion analysis: ten years' experiences and recent developments.

Since the implementation of the computer-aided system for assessing facial palsy in 1999 by Frey et al (Plast Reconstr Surg. 1999;104:2032-2039), no similar system that can make an objective, three-dimensional, quantitative analysis of facial movements has been marketed. This system has been in routine use since its launch, and it has proven to be reliable, clinically applicable, and therapeutically accurate. With the cooperation of international partners, more than 200 patients were analyzed. Recent developments in computer vision--mostly in the area of generative face models, applying active--appearance models (and extensions), optical flow, and video-tracking-have been successfully incorporated to automate the prototype system. Further market-ready development and a business partner will be needed to enable the production of this system to enhance clinical methodology in diagnostic and prognostic accuracy as a personalized therapy concept, leading to better results and higher quality of life for patients with impaired facial function.

Coenzyme Q10 does not enhance preadipocyte viability in an in vitro lipotransfer model.

BACKGROUND: Autologous fat is an attractive soft-tissue filler in plastic and reconstructive surgery. The success of the procedure relies strongly on the technique of transferring viable preadipocytes. Among other factors, preadipocyte viability is impaired by local anesthetics. Application of coenzyme Q10 is being performed by aesthetic plastic surgeons to enhance the success of lipotransfer. The aim of this study was to evaluate the effect of Q10 on preadipocyte viability with special regard to impairment after lidocaine treatment. METHODS: Preadipocytes were pretreated with coenzyme Q10 or vehicle control followed by incubation with lidocaine for 30 min. Viability and apoptosis were assessed by FACS analysis and Western blot. RESULTS: Coenzyme Q10 did not improve viability nor have any effect on investigated apoptosis parameters. Preadipocyte viability was reduced after lidocaine treatment. Surface binding of annexin V, cleavage of caspase-3, and abundance of subdiploid cells were not detectable though, suggesting that necrosis rather than apoptosis is the cause for reduced preadipocyte viability. CONCLUSION: Our results indicate that Q10 does not improve preadipocyte viability. Preadipocyte cell death induced by lidocaine is not caused by apoptosis but by necrosis, which cannot be prevented by coenzyme Q10. These findings should be taken into account when searching for solutions to improve preadipocyte viability in the context of soft tissue engineering and autologous fat transfer.

Biological effect and chemical monitoring of Watch List substances in European surface waters: Steroidal estrogens and diclofenac - Effect-based methods for monitoring frameworks.

Three steroidal estrogens, 17α-ethinylestradiol (EE2), 17ß-estradiol (E2), estrone (E1), and the non-steroidal anti-inflammatory drug (NSAID), diclofenac have been included in the first Watch List of the Water Framework Directive (WFD, EU Directive 2000/60/EC, EU Implementing Decision 2015/495). This triggered the need for more EU-wide surface water monitoring data on these micropollutants, before they can be considered for inclusion in the list of priority substances regularly monitored in aquatic ecosystems. The revision of the priority substance list of the WFD offers the opportunity to incorporate more holistic bioanalytical approaches, such as effect-based monitoring, alongside single substance chemical monitoring. Effect-based methods (EBMs) are able to measure total biological activities (e.g., estrogenic activity or cyxlooxygenase [COX]-inhibition) of specific group of substances (such as estrogens and NSAIDs) in the aquatic environment at low concentrations (pg/L). This makes them potential tools for a cost-effective and ecotoxicologically comprehensive water quality assessment. In parallel, the use of such methods could build a bridge from chemical status assessments towards ecological status assessments by adressing mixture effects for relevant modes of action. Our study aimed to assess the suitability of implementing EBMs in the WFD, by conducting a large-scale sampling and analysis campaign of more than 70 surface waters across Europe. This resulted in the generation of high-quality chemical and effect-based monitoring data for the selected Watch List substances. Overall, water samples contained low estrogenicity (0.01-1.3 ng E2-Equivalent/L) and a range of COX-inhibition activity similar to previously reported levels (12-1600 ng Diclofenac-Equivalent/L). Comparison between effect-based and conventional analytical chemical methods showed that the chemical analytical approach for steroidal estrogens resulted in more (76%) non-quantifiable data, i.e., concentrations were below detection limits, compared to the EBMs (28%). These results demonstrate the excellent and sensitive screening capability of EBMs.

Correlation of functional recovery with the course of electrophysiological parameters after free muscle transfer for reconstruction of the smile in irreversible facial palsy.

INTRODUCTION: The aim of this study was to determine whether processes of denervation and reinnervation, as measured by electrodiagnostic methods, correlate with clinical function, as measured by three-dimensional (3D) video analysis and whether electrodiagnostic data can serve as a prognostic indicator. METHODS: Eighteen patients with facial palsy were investigated by 3D video analysis, needle electromyography, and electrical muscle testing at 6, 12, and 18 months after free muscle transplantation for smile reconstruction. RESULTS: Electrophysiological parameters determined 6 months postoperatively correlated significantly with the index of dynamic symmetry 12 and 18 months postoperatively. CONCLUSIONS: Processes of reinnervation can be detected earlier by electrophysiological analysis than by quantified clinical analysis. Pathological spontaneous activity alone and combined assessment with motor unit action potentials in the early postoperative stage are strong prognostic indicators.

Influence of brachial plexus blockade on oxygen balance during surgery.

The combined effects of anesthesia, motor blockade, and chemically induced sympathectomy after brachial plexus blockade can have a beneficial impact, when applied in selected, isolated diseased states of the upper limb. With the aim of using the prolonged effects of brachial plexus blockade for a future therapeutic application, we demonstrated a dependable methodology of venous blood gas monitoring and confirmed an improved oxygen balance of the blocked versus nonblocked upper extremity in a controlled, prospective study in healthy patients undergoing elective hand surgery.

Electrical injury: a long-term analysis with review of regional differences.

Due to its relatively small share among burn injuries, published data on electrical injuries remain scarce, and differ in patient collectives due to infrastructural differences. We have retrospectively analyzed records of 56 patients who were admitted because of electrical injury to our burn center from 1994 to 2008, compared results with the current literature, and focused our review on regional differences. Patients in our collective were predominantly young men (71%, n = 40/56) and those who resulted from work-related accidents (59%, n = 33/56). The mean total burn surface area was 26%. In all, 93% of patients needed at least 1 operation, with 43% of patients requiring at least 1 surgical intervention during a follow-up hospital stay. The mean length-of-stay was 44 days. Two patients died, accounting for a mortality rate of 3.6%. When comparing high to low-voltage injuries, patients in the former group were significantly younger, had more operations, and required a longer length-of-stay. With respect to work-related high-voltage injuries, job-specific male-predominance explains for the demographic distribution of admissions. Low-voltage injuries continue to have low mortality rates in this part of Europe, most likely as a result of established high security standards as well as access to emergency treatment with subsequent intensive and specialist surgical care.

Adipose tissue engineering: three different approaches to seed preadipocytes on a collagen-elastin matrix.

BACKGROUND: Millions of plastic and reconstructive surgical procedures are performed each year to repair soft-tissue defects that result from significant burns, tumor resections, or congenital defects. Tissue-engineering strategies have been investigated to develop methods for generating soft-tissue. Preadipocytes represent a promising autologous cell source for adipose tissue engineering. These immature precursor cells, which are located between the mature adipocytes in the adipose tissue, are much more resistant to mechanical stress and ischemic conditions than mature adipocytes. To use preadipocytes for tissue-engineering purposes, cells were isolated from human adipose tissue and seeded onto scaffolds. Once processed, preadipocytes become subject to the human tissue act and require handling under much tighter regulations. Therefore, we intended to identify any influence caused by processing of preadipocytes prior to seeding on the reconstructed adipose tissue formation. MATERIAL AND METHOD: Human preadipocytes were isolated from subcutaneous adipose tissue obtained from discarded tissue during abdominoplasties of healthy men and women. Preadipocytes were divided into 3 groups. Cells of group I were seeded onto the scaffold directly after isolation, cells of group II were proliferated for 4 days before seeding, and cells of group III were proliferated and induced to differentiate before seeded onto the scaffold. A 3-dimensional scaffold (Matriderm, Dr. Otto Suwelack Skin and Health Care GmbH, Billerbeck, Germany) containing bovine collagen and elastin served as a carrier. Fourteen days after isolation, all scaffolds were histologically evaluated, using hematoxylin and eosin, anti-Ki-67 antibody, as well as immunofluorescence labeling with Pref-1 antibody (DLK (C-19), peroxisome proliferator-activated receptor gamma antibody, and DAPI (4',6-diamidino-2-phenylindole). RESULTS: Cells of all groups adhered to the scaffolds on day 21 after isolation. Cells of groups I (freshly isolated preadipocytes) and II (proliferated preadipocytes) adhered well and penetrated into deeper layers of the matrix. In group III (induced preadipocytes), penetration of cells was primarily observed to the surface area of the scaffold. DISCUSSION/CONCLUSION: : The collagen-elastin matrix serves as a useful scaffold for adipose tissue engineering. Freshly isolated preadipocytes as well as proliferated preadipocytes showed good penetration into deeper layers of the scaffold, whereas induced preadipocytes attached primarily to the surface of the matrix. We conclude that there might be different indications for each approach.

[Acute renale failure in burns - causes, consequences and avoidance strategies]. / Akutes Nierenversagen bei Brandverletzten - Ursachen, Konsequenzen und Vermeidungstrategien.

Acute renal failure (ARF) is a common and dangerous complication in intensive care medicine. Especially critical ill patients, who are suffering from major burns, have a high risk to develop ARF as a consequence of their trauma. Many factors, including the trauma itself, the damage of soft tissue and consecutive rhabdomyolysis, the development of the burn illness and therapeutic interventions play also a major role in this context. These circumstances have a major impact on the morbidity and mortality of severely burned patients. The aim of this manuscript is to review the reasons for the development of an ARF in burn patients as well as its consequences; moreover it highlights potential strategies to avoid ARF in critically ill burned patients.

Extraction With Sweat-Sebum Emulsion as a New Test Method for Leachables in Patch-Based Medical Devices, Illustrated by Assessment of Isobornylacrylate (IBOA) in Diabetes Products.

BACKGROUND: The increasing offering of patch-based medical devices is accompanied by growing numbers of reported adverse skin reactions. Procedures for testing leachables according to ISO 10993 may not be optimal for lipophilic substances that can be mobilized on skin by sweat and sebum. We propose an improved extraction method for targeted analysis of leachables using low volumes of a sweat-sebum emulsion. The approach is illustrated by the analysis of isobornylacrylate (IBOA), a compound found in some devices and suspected for allergenic potential. METHOD: Three patch-based products were tested: an implantable device for continuous glucose monitoring (CGM), an intermittently scanned CGM (isCGM) device, and a micro-insulin pump. Quantification of IBOA was performed by gas chromatography and allergenic potential of IBOA levels was assessed by the KeratinoSens cell assay. Different combinations were used for extraction solvent (isopropanol, 5% ethanol-water solution, and sweat-sebum emulsion), extraction volumes (complete immersion vs partial immersion in 2 mm of solvent), and extraction time (3, 5, and 14 days). RESULTS: Isobornylacrylate was only found in the isCGM device. About 20 mg/L IBOA were eluted after 3 days in isopropanol but only about 1 mg/L in ethanol-water. Sweat-sebum emulsion dissolves IBOA better and gives a more stable solution than ethanol-water. Decomposition of IBOA solutions requires adjusted extraction timing or correction of results. In the sweat-sebum extract, IBOA levels were about 20 mg/L after 3 days and about 30 mg/L after 5 days, clearly above the threshold found in the KerationSens assay for keratinocyte activation (10 mg/L). CONCLUSION: Extraction by low volumes of sweat-sebum emulsion can be a superior alternative for the targeted simulating-use assessment of leachables in patch-based medical devices.

Long-term outcomes of web creep, scar quality, and function after simple syndactyly surgical treatment.

PURPOSE: Syndactyly is the second most common congenital malformation of the hand, and reports of the incidence of web creep after surgery vary. To evaluate our outcomes of simple syndactyly surgical release, we conducted a retrospective analysis of patients treated between January 1965 and December 2007. METHODS: After matching for inclusion criteria, we recruited 19 patients with 26 affected web spaces for clinical examination. Outcomes evaluation included grading of web creep, Vancouver Scar Scale, assessment of complications and subjective patient analysis, range of motion, degree of finger abduction, power, and 2-point discrimination. Mean age at follow-up was 18 years (range, 6-50 y), with a mean age of 4.4 years (range, 7 mo to 15 y) at surgery and mean follow-up of 11.5 years (range, 5-35 y). Surgical management consisted of palmar and dorsal triangular skin flaps for creation of the new commissure, and multiple zigzag incisions for separation of digits. For tension-free closure, full-thickness skin grafts were harvested as needed. RESULTS: We observed web creep up to the proximal third of the distance between palmar metacarpophalangeal and proximal interphalangeal joint crease in 2 web spaces. All other web spaces had either a soft web equivalent to the contralateral (unaffected) side (n = 13) or no web advancement with thickening of the interdigital space (n = 11). The scar quality as assessed with the Vancouver Scar Scale revealed a height below 2 mm in 24 of 26 web spaces, with close to normal to supple pliability in 20 of 26 web spaces. There were no considerable differences for range of motion, degree of finger abduction, power, or 2-point discrimination between the affected and unaffected sides. In 17 of 24 cases in which full-thickness skin grafts from the groin region were used, patients reported commissural hair growth in the grafted region. CONCLUSIONS: Evaluation of the long-term outcomes of surgical treatment for simple syndactyly at our institution demonstrated a low incidence of web creep. When choosing the groin as a donor area for full thickness skin grafts, we recommend harvesting from the lateral third of the inguinal crease, to avoid esthetic compromise associated with the beginning of hair growth in puberty. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Pathophysiology of burns.

Burn injury represents a significant problem worldwide. Advances in therapy strategies, based on better understanding of the pathophysiologic responses after burn injury have improved the clinical outcome of patients with burn injuries over the past years. This article describes the present understanding of the pathophysiology of a burn injury including both the local and systemic responses, focusing on the many facets of organ and systemic effects directly resulting from hypovolemia and circulating mediators following burn trauma.

Clinical application of pectoral nerve transfers in the treatment of traumatic brachial plexus injuries.

PURPOSE: To determine the effectiveness, reliability and donor site morbidity of pectoral nerve transfers in the treatment of brachial plexus lesions. METHODS: This retrospective study included 6 patients with 7 pectoral nerve transfers. The follow-up examination comprised measurement of the ranges of motion (AO neutral-0 method), functional muscle testing (British Medical Research Council), and photo- and videographic documentation. RESULTS: Three patients achieved excellent results (M5, M4), in 3 cases good muscle function was achieved (M3) and 1 patient, who had a short follow-up period, revealed M2 motor function. CONCLUSIONS: This study describes the treatment of patients with conditions far from ideal. All patients sustained extensive brachial plexus lesions and most had severe associated injuries of the upper limb, which limited the availability of sufficient donor nerves. Despite these facts, 6 cases had excellent or good results and 1 showed continuing improvement of muscle function. These good results and the excellent results documented in literature demonstrate the high dependability and efficiency of the pectoral nerves as donors in the treatment of brachial plexus lesions. Furthermore, it was shown that free functioning muscle transplants can be sufficiently reinnervated by pectoral nerves. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Two novel real time cell-based assays quantify beta-blocker and NSAID specific effects in effluents of municipal wastewater treatment plants.

Pharmaceuticals, such as beta-blockers, nonsteroidal anti-inflammatory drugs (NSAIDs) as well as their metabolites are introduced into the water cycle via municipal wastewater treatment plant (WWTP) effluents in all industrialized countries. As the amino acid sequences of the biological target molecules of these pharmaceuticals - the beta-1 adrenergic receptor for beta-blockers and the cyclooxygenase for NSAIDs - are phylogenetically conserved among vertebrates it is reasonable that wildlife vertebrates including fish physiologically respond in a similar way to them as documented in humans. Consequently, beta-blockers and NSAIDs both exhibit their effects according to their mode of action on one hand, but on the other hand that may lead to unwanted side effects in non-target species. To determine whether residuals of beta-1 adrenergic receptor antagonists and cyclooxygenase inhibitors may pose a risk to aquatic organisms, one has to know the extent to which such organisms respond to the total of active compounds, their metabolites and transformation products with the same modes of action. To cope with this demand, two cell-based assays were developed, by which the total beta-blocker and cyclooxygenase inhibitory activity can be assessed in a given wastewater or surface water extract in real time. The measured activity is quantified as metoprolol equivalents (MetEQ) of the lead substance metoprolol in the beta-blocker assay, and diclofenac equivalents (DicEQ) in the NSAID assay. Even though MetEQs and DicEQs were found to surpass the concentration of the respective lead substances (metoprolol, diclofenac), as determined by chemical analysis by a factor of two to three, this difference was shown to be reasonably explained by the presence and action of additional active compounds with the same mode of action in the test samples. Thus, both in vitro assays were proven to integrate effectively over beta-blocker and NSAID activities in WWTP effluents in a very sensitive and extremely rapid manner.

Worldwide Testing of the eFACE Facial Nerve Clinician-Graded Scale.

BACKGROUND: The electronic, clinician-graded facial function scale (eFACE) is a potentially useful tool for assessing facial function. Beneficial features include its digital nature, use of visual analogue scales, and provision of graphic outputs and scores. The authors introduced the instrument to experienced facial nerve clinicians for feedback, and examined the effect of viewing a video tutorial on score agreement. METHODS: Videos of 30 patients with facial palsy were embedded in an Apple eFACE application. Facial nerve clinicians were invited to perform eFACE video rating and tutorial observation. Participants downloaded the application, viewed the clips, and applied the scoring. They then viewed the tutorial and rescored the clips. Analysis of mean, standard deviation, and confidence interval were performed. Values were compared before and after tutorial viewing, and against scores obtained by an experienced eFACE user. RESULTS: eFACE feedback was positive; participants reported eagerness to apply the instrument in clinical practice. Standard deviation decreased significantly in only two of the 16 categories after tutorial viewing. Subscores for static, dynamic, and synkinesis all demonstrated stable standard deviations, suggesting that the instrument is intuitive. Participants achieved posttutorial scores closer to the experienced eFACE user in 14 of 16 scores, although only a single score, nasolabial fold orientation with smiling, achieved statistically significant improvement. CONCLUSIONS: The eFACE may be a suitable, cross-platform, digital instrument for facial function assessment, and was well received by facial nerve experts. Tutorial viewing does not appear to be necessary to achieve agreement, although it does mildly improve agreement between occasional and frequent eFACE users.