Cell Competition: Mechanisms and Physiological Roles.

Cell-competitive interactions are widespread in nature and determine the outcome of a vast variety of biological processes. A particular class of competitive interactions takes place when alterations in intrinsic cellular properties are sensed nonautonomously by comparison between neighboring cells, resulting in the selective elimination of one cell population. This type of cell competition was first described four decades ago in developing epithelia of Drosophila. In the last 15 years, further molecular and cellular analyses have provided essential knowledge about the mechanisms, universality, and physiological relevance of cell competition. The two main phenomena triggering cell competition are alterations in cellular metabolic status and alterations in epithelial apico-basal polarity, while other reported pathways are less characterized. Cell competition plays essential roles in quality control, homeostasis, and repair of developing and adult tissues, and depending on the context, it may function as a tumor-suppressing or tumor-promoting mechanism.

Visceral adipose tissue quality was associated with nonalcoholic fatty liver disease, independent of its quantity.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is a serious liver disease. Recent studies have shown that both visceral adipose tissue (VAT) quantity and density (as an indirect measure of quality) are associated with metabolic profiles. Therefore, we investigated the association between VAT quantity and quality, and the prevalence and incidence of NAFLD. METHODS AND RESULTS: In this cross-sectional, retrospective cohort study, the prevalence and incidence of NAFLD were analyzed in 627 and 360 middle-aged subjects, respectively. VAT was evaluated using an unenhanced computed tomography scan, while NAFLD was evaluated using ultrasonography. The VAT area was normalized to the square value of the subjects' height in meters, the visceral fat area (VFA) index. The VAT density was described as the visceral fat density (VFD). The VFA index and VFD had an interaction effect on the prevalence of NAFLD (P = 0.0059). The VFA index (adjusted odds ratio [OR], 1.04; 95% confidence interval [CI], 1.01-1.07; P = 0.0145, per 1.0 cm2/m2) and the VFD (OR, 0.90; 95% CI, 0.84-0.96; P = 0.0026, per 1.0 Hounsfield unit [HU]) were independently associated with the prevalence of NAFLD. In our cohort, 36 subjects developed NAFLD. The VFD (adjusted hazards ratio [HR], 0.84; 95% CI, 0.77-0.91; P < 0.0001, per 1.0 HU) was independently associated with the incidence of NAFLD, whereas the VFA index was not. CONCLUSION: Both the VFA index and VFD were independently associated with NAFLD prevalence. The VFD might be more related to the incidence of NAFLD than the VFA index.

Rapid and sensitive mycoplasma detection system using image-based deep learning.

A major concern in the clinical application of cell therapy is the manufacturing cost of cell products, which mainly depends on quality control. The mycoplasma test, an important biological test in cell therapy, takes several weeks to detect a microorganism and is extremely expensive. Furthermore, the manual detection of mycoplasma from images requires high-level expertise. We hypothesized that a mycoplasma identification program using a convolutional neural network could reduce the test time and improve sensitivity. To this end, we developed a program comprising three parts (mycoplasma detection, prediction, and cell counting) that allows users to evaluate the sample and verify infected/non-infected cells identified by the program. In experiments conducted, stained DNA images of positive and negative control using mycoplasma-infected and non-infected Vero cells, respectively, were used as training data, and the program results were compared with those of conventional methods, such as manual counting based on visual observation. The minimum detectable mycoplasma contaminations for manual counting and the proposed program were 10 and 5 CFU (colony-forming unit), respectively, and the test time for manual counting was 20 times that for the proposed program. These results suggest that the proposed system can realize a low-cost and streamlined manufacturing process for cellular products in cell-based research and clinical applications.

Correlation of cerebellar granular layer autolysis with ante-mortem systemic acid-base status.

Research in neuroscience relies heavily upon postmortem human brain tissue. Cerebellar granular layer autolysis (GLA) is a surrogate marker for the quality of such tissue and suitability for molecular analysis. GLA is associated with reduced brain tissue pH. The aim of this study was to assess correlation of GLA with premortem systemic acid-base status. This is a retrospective study in which 62 consecutive adult autopsy cases were included. Sections of cerebellum were reviewed microscopically for presence of GLA. Autolysis was graded as negative, grade 1, grade 2, and grade 3. Medical records were reviewed for arterial blood gas analysis. Postmortem interval was recorded. 23 of 62 cases showed GLA. Of the 23 patients with autolysis, 22 were acidotic and 1 was alkalotic. Of these 23 cases, 15 had metabolic acidosis, 4 had respiratory acidosis, 3 had combined acidosis and 1 had respiratory alkalosis. There was no statistically significant difference in postmortem interval between the two groups. 10 cases with grade 3 autolysis had mean pH of 7.13, 7 cases with grade 2 autolysis had mean pH of 7.23 and in 6 cases with grade 1 autolysis the mean pH was 7.2. Overall, the mean pH in patients with GLA was 7.19, and in the non-autolytic cases the mean pH was 7.28 (P < 0.05). There was no correlation between the degree of acidosis and severity of autolysis. GLA is associated with premortem systemic acidosis, and premortem systemic alkalosis is associated with the absence of GLA. Premortem acid-base status may serve as an additional quality indicator for assessment of tissue for research.

[The research of the connective tissue dysplasia effect on dental eruption and hard tissues mineralization]. / Vliyanie displazii soedinitel'noi tkani na sroki prorezyvaniya i stepen' mineralizatsii zubov cheloveka.

OBJECTIVE: The purpose of this investigation was to explore the quality of 38, 48 teeth's hard tissues at different eruption stages and mandibular bone in different postpartum ontogenesis periods. MATERIAL AND METHODS: The research involved 102 male patients divided into groups according to their age: 15-20, 21-30 years old, they were extracted one tooth 38, 48 without inflammatory process signs and a fragment of the mandibular bone alveolar part in the projection of the teeth 38, 48 for orthodontic indications. In the comparison group (49 observations without signs of connective tissue dysplasia), in the study group (53 observations with signs of connective tissue dysplasia) we extracted teeth 38, 48 which were in the bone tissue. We analyzed condition of crown and root systems of extracted 38, 48 teeth, densitometric density of mineral component, size of enamel prisms, size of prismatic shells of organic matrix, spatial organization of collagen fibers in bone tissue, size characteristics of bone plates and mineralization centers of bone tissue. Processing of the obtained data was performed by methods of variation statistics using standard packages Microsoft Excel 2008, Statistica 12.0. RESULTS: The paper reveals one of the surgical dentistry pressing issues related to the tething mechanism of lower human wisdom teeth, considered by the authors from the position of tissue disorders in collagen type 1 observed in connective tissue dysplasia. At age of 15-20 years in connective tissue dysplasia, the enamel prisms hypomineralized areas are generalized; at age of 21-30 years the hypomineralized areas are characterized by local changes. At the age of 15-20 years in connective tissue dysplasia the bone plates splitting is observed at the level of most fibrils, collagen fibers have insufficiently oriented direction in contrast to the age group of 21-30 years where bone plates splitting is characteristic for single fibrils and collagen fibrils are clearly oriented. CONCLUSION: Morphological and histological changes in teeth 38, 48 and in bone tissue prevent correct and timely teething at 15-20 years and create more favorable conditions for teething at 21-30 years with a slower rate in connective tissue dysplasia, but in both groups teeth eruption occurs under unfavorable anatomic conditions.

Characterization of PD-L1 expression in Chinese non-small cell lung cancer patients with PTEN expression as a means for tissue quality screening.

The goal of this study is to evaluate PD-L1 prevalence and its association with major clinical characteristics in Chinese non-small cell lung cancer (NSCLC) patients to inform the clinical development of anti-PD1/PD-L1 agents in this population. We used phosphatase and tensin homolog (PTEN) expression through IHC as a surrogate tissue quality marker to screen surgical NSCLC samples in tissue microarray (TMA; 172 cases) or whole-section (268 cases) format. The samples were then analyzed with a clinically validated PD-L1 IHC assay. The results were correlated with baseline characteristics and clinical outcomes. PTEN IHC showed that 108 TMA samples and 105 whole-section samples qualified for PD-L1 IHC. With a clinically relevant cutoff, 41.7% of the TMA samples were PD-L1 positive. PD-L1 level was much lower in EGFR-mutant patients and seemed to be a favorable prognostic factor for both overall survival (OS) and recurrence-free survival (RFS). These findings were confirmed in the whole-section samples except that their survival data were not mature enough for correlation analysis. In summary, PD-L1 expression was detected in approximately 40% of PTEN-qualified Chinese NSCLC samples, negatively correlated with EGFR mutation and seemed to be a favorable prognostic factor for both OS and RFS. Notably, the different results from PTEN-qualified and PTEN-disqualified samples underscore the importance of tissue quality control prior to biomarker testing.

Indications and graft survival analysis in optical penetrating keratoplasty in a tertiary care center in North India: a 5-year study.

PURPOSE: To study the indications and variables correlating with graft survival in optical penetrating keratoplasty in a tertiary care hospital in north India. PARTICIPANTS: All patients who underwent optical keratoplasty, except those undergoing lamellar grafts, tectonic grafts transplants and penetrating keratoplasty done for therapeutic purposes were included in the study. Patients with follow-up less than 2 years were excluded from the study. MATERIALS AND METHODS: Data were obtained by reviewing the records of 101 patients who underwent optical penetrating keratoplasty from 2008 to 2013 for various indications. RESULTS: Out of 101 patients who underwent optical penetrating keratoplasty, 71 were males and 30 were females. The mean age of the recipient was 48.53 years with range 1-82 years. The main indications were previous failed graft (29.7%), healed keratitis except HSV (15.8%), pseudophakic or aphakic bullous keratopathy (14.8%), corneal dystrophies/degenerations (12.9%), adherent leucoma (9.9%), post-HSV scars (8.9%), and others like anterior staphyloma, congenital corneal opacities, buphthalmos and keratoconus (7.9%). The graft survival rate was 67.33% at 1-year follow-up and 59.4% at 2-year follow-up. The mean survival time of the grafts was 22.42 months. The mean time for graft failure was 7.12 (±0.9) months. CONCLUSION: One- and 2-year survival of grafts at our center is lower as compared to western studies, probably due to higher percentage of poor prognosis indications for surgery and a relative scarcity of excellent-quality donor corneas.

Multicenter fresh frozen tissue sampling in colorectal cancer: does the quality meet the standards for state of the art biomarker research?

The growing interest in the molecular subclassification of colorectal cancers is increasingly facilitated by large multicenter biobanking initiatives. The quality of tissue sampling is pivotal for successful translational research. This study shows the quality of fresh frozen tissue sampling within a multicenter cohort study for colorectal cancer (CRC) patients. Each of the seven participating hospitals randomly contributed ten tissue samples, which were collected following Standard Operating Procedures (SOP) using established techniques. To indicate if the amount of intact RNA is sufficient for molecular discovery research and prove SOP compliance, the RNA integrity number (RIN) was determined. Samples with a RIN < 6 were measured a second time and when consistently low a third time. The highest RIN was used for further analysis. 91% of the tissue samples had a RIN ≥ 6 (91%). The remaining six samples had a RIN between 5 and 6 (4.5%) or lower than 5 (4.5%). The median overall RIN was 7.3 (range 2.9-9.0). The median RIN of samples in the university hospital homing the biobank was 7.7 and the median RIN for the teaching hospitals was 7.3, ranging from 6.5 to 7.8. No differences were found in the outcome of different hospitals (p = 0.39). This study shows that the collection of high quality fresh frozen samples of colorectal cancers is feasible in a multicenter design with complete SOP adherence. Thus, using basic sampling techniques large patient cohorts can be organized for predictive and prognostic (bio)marker research for CRC.

Mass spectrometric characterization of limited proteolysis activity in human plasma samples under mild acidic conditions.

We developed a limited proteolysis assay for estimating dynamics in plasma-borne protease activities using MALDI ToF MS analysis as readout. A highly specific limited proteolysis activity was elicited in human plasma by shifting the pH to 6. Mass spectrometry showed that two singly charged ion signals at m/z 2753.44 and m/z 2937.56 significantly increased in abundance under mild acidic conditions as a function of incubation time. For proving that a provoked proteolytic activity in mild acidic solution caused the appearance of the observed peptides, control measurements were performed (i) with pepstatin as protease inhibitor, (ii) with heat-denatured samples, (iii) at pH 1.7, and (iv) at pH 7.5. Mass spectrometric fragmentation analysis showed that the observed peptides encompass the amino acid sequences 1-24 and 1-26 from the N-terminus of human serum albumin. Investigations on peptidase specificities suggest that the two best candidates for the observed serum albumin cleavages are cathepsin D and E. Reproducibility, robustness, and sensitivity prove the potential of the developed limited proteolysis assay to become of clinical importance for estimating dynamics of plasma-borne proteases with respect to associated pathophysiological tissue conditions.

New insights to the role of aryl hydrocarbon receptor in bone phenotype and in dioxin-induced modulation of bone microarchitecture and material properties.

Bone is a target for high affinity aryl hydrocarbon receptor (AHR) ligands, such as dioxins. Although bone morphology, mineral density and strength are sensitive endpoints of dioxin toxicity, less is known about effects on bone microarchitecture and material properties. This study characterizes TCDD-induced modulations of bone tissue, and the role of AHR in dioxin-induced bone toxicity and for normal bone phenotype. Six AHR-knockout (Ahr(-/-)) and wild-type (Ahr(+/+)) mice of both genders were exposed to TCDD weekly for 10 weeks, at a total dose of 200µg/kgbw. Bones were examined with micro-computed tomography, nanoindentation and biomechanical testing. Serum levels of bone remodeling markers were analyzed, and the expression of genes related to osteogenic differentiation was profiled using PCR array. In Ahr(+/+) mice, TCDD-exposure resulted in harder bone matrix, thinner and more porous cortical bone, and a more compact trabecular bone compartment. Bone remodeling markers and altered expression of a number of osteogenesis related genes indicated imbalanced bone remodeling. Untreated Ahr(-/-) mice displayed a slightly modified bone phenotype as compared with untreated Ahr(+/+) mice, while TCDD exposure caused only a few changes in bones of Ahr(-/-) mice. Part of the effects of both TCDD-exposure and AHR-deficiency were gender dependent. In conclusion, exposure of adult mice to TCDD resulted in harder bone matrix, thinner cortical bone, mechanically weaker bones and most notably, increased trabecular bone volume fraction in Ahr(+/+) mice. AHR is involved in bone development of a normal bone phenotype, and is crucial for manifestation of TCDD-induced bone alterations.

Ethanol, Carnoy, and paraformaldehyde as fixative solutions for histological evaluation of preantral follicles in equine ovarian tissue.

The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy's (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters in equine ovarian tissue. Ovaries (n = 6) from ovariectomized young mares were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1444 histological slides. The ST-EtOH similarly preserved the preantral follicle morphometry and stromal cell density compared to the PFA fixative, regardless of the exposure time. Nonetheless, the CAR fixative solution had the greatest percentage of normal preantral follicles and the highest stromal cell density among all treatments. In conclusion, Carnoy's solution must be preferred compared with ST-EtOH and PFA fixatives for studies concerning the cellular morphology of equine ovarian tissue. Moreover, ST-EtOH fixative is a good alternative for equine ovarian tissue when a quick histological evaluation is required instead of more time-consuming and expensive techniques. Additional studies concerning the impact of different fixatives on the ultrastructure of cellular populations and their compatibility with IHC and molecular techniques in equine ovarian tissue are warranted.

Raman Spectroscopy in Skeletal Tissue Disorders and Tissue Engineering: Present and Prospective.

Musculoskeletal disorders are the most common reason of chronic pain and disability, representing an enormous socioeconomic burden worldwide. In this review, new biomedical application fields for Raman spectroscopy (RS) technique related to skeletal tissues are discussed, showing that it can provide a comprehensive profile of tissue composition in situ, in a rapid, label-free, and nondestructive manner. RS can be used as a tool to study tissue alterations associated to aging, pathologies, and disease treatments. The main advantage with respect to currently applied methods in clinics is its ability to provide specific information on molecular composition, which goes beyond other diagnostic tools. Being compatible with water, RS can be performed without pretreatment on unfixed, hydrated tissue samples, without any labeling and chemical fixation used in histochemical methods. This review first provides the description of the basic principles of RS as a biotechnology tool and is introduced into the field of currently available RS-based techniques, developed to enhance Raman signals. The main spectral processing, statistical tools, fingerprint identification, and available databases are mentioned. The recent literature has been analyzed for such applications of RS as tendon and ligaments, cartilage, bone, and tissue engineered constructs for regenerative medicine. Several cases of proof-of-concept preclinical studies have been described. Finally, advantages, limitations, future perspectives, and challenges for the translation of RS into clinical practice have been also discussed. Impact statement Raman spectroscopy (RS) is a powerful noninvasive tool giving access to molecular vibrations and characteristics of samples in a wavelength window of 600 to 3200 cm-1, thus giving access to a molecular fingerprint of biological samples in a nondestructive way. RS could not only be used in clinical diagnostics, but also be used for quality control of tissues and tissue-engineered constructs, reducing number of samples, time, and the variety of analysis required in the quality control chain before implantation.

How to learn with intentional mistakes: NoisyEnsembles to overcome poor tissue quality for deep learning in computational pathology.

There is a lot of recent interest in the field of computational pathology, as many algorithms are introduced to detect, for example, cancer lesions or molecular features. However, there is a large gap between artificial intelligence (AI) technology and practice, since only a small fraction of the applications is used in routine diagnostics. The main problems are the transferability of convolutional neural network (CNN) models to data from other sources and the identification of uncertain predictions. The role of tissue quality itself is also largely unknown. Here, we demonstrated that samples of the TCGA ovarian cancer (TCGA-OV) dataset from different tissue sources have different quality characteristics and that CNN performance is linked to this property. CNNs performed best on high-quality data. Quality control tools were partially able to identify low-quality tiles, but their use did not increase the performance of the trained CNNs. Furthermore, we trained NoisyEnsembles by introducing label noise during training. These NoisyEnsembles could improve CNN performance for low-quality, unknown datasets. Moreover, the performance increases as the ensemble become more consistent, suggesting that incorrect predictions could be discarded efficiently to avoid wrong diagnostic decisions.

PD-L1 expression in head and neck cancer tissue specimens decreases with time.

BACKGROUND: PD-L1 immunohistochemical expression is used as an important theranostic marker in various malignancies, including head and neck squamous cell carcinoma (HNSCC) where the combined positive score (CPS) guides treatment decisions. Despite indirect evidence that there is loss of antigenicity for archived tissues, there is no direct comparison between PD-L1 expression of the same tissue upon arrival and after its storage. MATERIAL AND METHODS: We compared the immunohistochemical expression of PD-L1 (22C3) in 106 HNSCC upon their arrival and after their storage (interval ranging from 20 to 48 months, mean 30.8 months). The evaluation was performed by two different pathologists' groups. RESULTS: We found a statistically significant decrease in the PD-L1 tumor proportional score (TPS), immune cells expression (IC) and CPS between the initial and the newly stained slides. CONCLUSION: This is the first study comparing PD-L1 expression between a tissue and "himself" later in time, highlighting an important decrease in expression by tumor and immune cells, and suggesting that an immediate rather than a retrospective assay of PD-L1 expression should be preferable in the routine practice. DATA AVAILABILITY: Data are available upon reasonable request.

Presurgical tear characteristics and estimated shear modulus as predictors of repair integrity and shoulder function one year after rotator cuff repair.

BACKGROUND: Rotator cuff repair provides pain relief for many patients; however, retears are relatively common and affect approximately 20%-70% of patients after repair. Although magnetic resonance imaging (MRI) offers the ability to assess tissue characteristics such as tear size, retraction, and fatty infiltration, it provides little insight into the quality of the musculotendinous tissues the surgeon will encounter during surgery. However, shear wave elastography (SWE) could provide an indirect assessment of quality (ie, stiffness) by measuring the speed of shear waves propagating through tissue. The objective of this study was to determine the extent to which estimated shear modulus predicts repair integrity and functional outcomes 1 year after rotator cuff repair. METHODS: Thirty-three individuals scheduled to undergo arthroscopic rotator cuff repair were enrolled in this study. Before surgery, shear modulus of the supraspinatus tendon and muscle was estimated using ultrasound SWE. MRIs were obtained before and 1 year after surgery to assess tear characteristics and repair integrity, respectively. Shoulder strength, range of motion, and patient-reported pain and function were assessed before and after surgery. Functional outcomes were compared between groups and across time using a two-factor mixed model analysis of variance. Stepwise regression with model comparison was used to investigate the extent to which MRI and shear modulus predicted repair integrity and function at 1 year after surgery. RESULTS: At 1 year after surgery, 56.5% of patients had an intact repair. No significant differences were found in any demographic variable, presurgical tear characteristic, or shear modulus between patients with an intact repair and those with a recurrent tear. Compared with presurgical measures, patients in both groups demonstrated significant improvements at 1 year after surgery in pain (P < .01), self-reported function (P < .01), range of motion (P < .01), and shoulder strength (P < .01). In addition, neither presurgical MRI variables (P > .16) nor shear modulus (P > .52) was significantly different between groups at 1 year after surgery. Finally, presurgical shear modulus generally did not improve the prediction of functional outcomes above and beyond that provided by MRI variables alone (P > .22). CONCLUSION: Although SWE remains a promising modality for many clinical applications, this study found that SWE-estimated shear modulus did not predict repair integrity or functional outcomes at 1 year after surgery, nor did it add to the prediction of outcomes above and beyond that provided by traditional presurgical MRI measures of tear characteristics. Therefore, it appears that further research is needed to fully understand the clinical utility of SWE for musculoskeletal tissue and its potential use for predicting outcomes after surgical rotator cuff repair.

Application of CRISPR screens to investigate mammalian cell competition.

Cell competition is defined as the context-dependent elimination of cells that is mediated by intercellular communication, such as paracrine or contact-dependent cell signaling, and/or mechanical stresses. It is considered to be a quality control mechanism that facilitates the removal of suboptimal cells from both adult and embryonic tissues. Cell competition, however, can also be hijacked by transformed cells to acquire a 'super-competitor' status and outcompete the normal epithelium to establish a precancerous field. To date, many genetic drivers of cell competition have been identified predominately through studies in Drosophila. Especially during the last couple of years, ethylmethanesulfonate-based genetic screens have been instrumental to our understanding of the molecular regulators behind some of the most common competition mechanisms in Drosophila, namely competition due to impaired ribosomal function (or anabolism) and mechanical sensitivity. Despite recent findings in Drosophila and in mammalian models of cell competition, the drivers of mammalian cell competition remain largely elusive. Since the discovery of CRISPR/Cas9, its use in functional genomics has been indispensable to uncover novel cancer vulnerabilities. We envision that CRISPR/Cas9 screens will enable systematic, genome-scale probing of mammalian cell competition to discover novel mutations that not only trigger cell competition but also identify novel molecular components that are essential for the recognition and elimination of less fit cells. In this review, we summarize recent contributions that further our understanding of the molecular mechanisms of cell competition by genetic screening in Drosophila, and provide our perspective on how similar and novel screening strategies made possible by whole-genome CRISPR/Cas9 screening can advance our understanding of mammalian cell competition in the future.

Harnessing Endophytic Fungi for Enhancing Growth, Tolerance and Quality of Rose-Scented Geranium (Pelargonium graveolens (L'Hér) Thunb.) Plants under Cadmium Stress: A Biochemical Study.

Heavy metal contamination in soil is increasing rapidly due to increasing anthropogenic activities. Despite the importance of rose-scented geranium as a medicinal plant, little attention was paid to enhancing its productivity in heavy metal-polluted soil. In this regard, endophytes improve plant resistance to heavy metal toxicity and enhance its tissue quality. Here, the impact of the three endophytic fungi Talaromyces versatilis (E6651), Emericella nidulans (E6658), and Aspergillus niger (E6657) on geranium growth, tolerance, and tissue quality under cadmium (Cd) stress was investigated. In contrast to E. nidulans, T. versatilis and A. niger enhanced geranium growth and the stimulatory effect was more pronounced under Cd-stress. The three endophytes significantly alleviated Cd accumulation and increased mineral content in geranium leaves. In addition, endophytic fungi successfully alleviated Cd-induced membrane damage and reinforced the antioxidant defenses in geranium leaves. Inoculation with endophytes stimulated all the antioxidant enzymes under Cd-stress, and the response was more obvious in the case of T. versatilis and A. niger. To reduce the toxicity of tissue-Cd levels, T. versatilis and A. niger upregulated the detoxification mechanisms; glutathione-S-transferase, phytochelatin, and metallothionein levels. Moreover, endophytic fungi improved the medicinal value and quality of geranium by increasing total antioxidant capacity (TAC), phenolic compound biosynthesis (phenylalanine ammonia-lyase), and vitamin content as well as the quantity and quality of essential oil, particularly under Cd-stress conditions. The variation in the mechanisms modulated by the different endophytic fungi was supported by Principal Component Analysis (PCA). Overall, this study provided fundamental insights into endophytes' impact as a feasible strategy to mitigate the phytotoxicity hazards of Cd-stress in geranium and enhance its quality, based on the growth and biochemical investigations.

Biomechanical Changes of Repair Tissue after Autologous Chondrocyte Implantation at Long-Term Follow-Up.

Objective. This study aims to describe biomechanical maturation process of repair tissue after cartilage repair with autologous chondrocyte implantation (ACI) at long-term follow-up. Design. After ACI, 40 patients underwent altogether 60 arthroscopic biomechanical measurements of the repair tissue at various time points during an up to 11-year follow-up period. Of these patients, 30 patients had full-thickness cartilage lesions and 10 had an osteochondritis dissecans (OCD) defect. The mean lesion area was 6.5 cm2 (SD 3.2). A relative indentation stiffness value for each individually measured lesion was calculated as a ratio of repair tissue and surrounding cartilage indentation value to enable interindividual comparison. Results. Repair tissue stiffness improved during approximately 5 years after surgery. Most of the increase in stiffness occurred during the first 2 years. The curvilinear correlation between relative stiffness values and the follow-up time was 0.31 (95% CI 0.07-0.52), P = 0.017. The interindividual variation of the stiffness was high. Lesion properties or demographic factors showed no significant correlation to biomechanical outcome. The overall postoperative average relative stiffness was 0.75 (SD 0.47). Conclusions. Our clinical study describes a biomechanical maturation process of cartilage repair that may continue even longer than expected. A substantial increase in tissue stiffness proceeds for the first two years postoperatively. Minor progression proceeds for even longer. In some repairs, the biomechanical result was equal to native cartilage, suggesting hyaline-type repair. The variation in biomechanical results suggests substantial inconsistency in the structural outcome following ACI.

The extremely low energy cost of biosynthesis in holometabolous insect larvae.

The metabolic cost of growth, which quantifies the amount of energy required to synthesize a unit of biomass, is an important component of an animal's ontogenetic energy budget. Here we investigated this quantity as well as other energy budget variables of the larvae of a holometabolous insect species, Vanessa cardui (painted lady). We found that the high growth rate of this caterpillar cannot be explained by its metabolic rate and the percentage of the metabolic energy allocated to growth; the key to understanding its fast growth is the extremely low cost of growth, 336 Joules/gram of dry mass. The metabolic cost of growth in caterpillars is 15-65 times lower than that of the endothermic and ectothermic species investigated in previous studies. Our results suggest that the low cost cannot be attributed to its body composition, diet composition, or body size. To explain the "cheap price" of growth in caterpillars, we assumed that a high metabolic cost for biosynthesis resulted in a high "quality" of cells, which have fewer errors during biosynthesis and higher resistance to stressors. Considering the life history of the caterpillars, i.e., tissue disintegration during metamorphosis and a short developmental period and lifespan, we hypothesized that an energy budget that allocates a large amount of energy to biosynthesizing high quality cells would be selected against in this species. As a preliminary test of this hypothesis, we estimated the metabolic cost of growth in larvae of Manduca sexta (tobacco hornworm) and nymphs of Blatta lateralis (Turkestan cockroach). The preliminary data supported our hypothesis.

Effects of Alterations of Post-Mortem Delay and Other Tissue-Collection Variables on Metabolite Levels in Human and Rat Brain.

The use of post-mortem human tissue is indispensable in studies investigating alterations in metabolite levels in neurodegenerative conditions such as Alzheimer's disease (AD). However, variability between samples may have unknown effects on metabolite concentrations. The aim of this study was to characterize the impact of such variables. Cingulate gyrus was obtained from AD cases and controls, from three brain banks. Gas chromatography-mass spectrometry (GC-MS) was used to measure and compare the levels of 66 identifiable metabolites in these tissues to determine effects of tissue-collection variables. The effect of PMD was further investigated by analysis of rat brain cortex and cerebellum collected following post-mortem delays (PMDs) of zero to 72 h. Metabolite levels between cases and controls were not replicable across cohorts with variable age- and gender-matching, PMD, and control Braak staging. Analysis of rat tissues found significant effects of PMD on 31 of 63 identified metabolites over periods up to 72 h. PMD must be kept under 24 h for metabolomics analyses on brain tissues to yield replicable results. Tissues should also be well age- and gender-matched, and Braak stage in controls should be kept to a minimum in order to minimize the impact of these variables in influencing metabolite variability.