Effect of Trehalose Supplementation in Egg-Yolk-Free Extender on Conventional Parameters and Gene Expression Related to Reactive Oxygen Species, Apoptosis, and Motility of Frozen Dog Spermatozoa.

The present study was conducted to evaluate the effects of trehalose supplementation in egg-yolk (EY)-free tris extender on dog spermatozoa. Pooled spermatozoa were diluted with extender 1 (EY-free tris extender supplemented with 0, 10, 15, 20, or 30 mM trehalose) and cooled (2 × 108 sperm/mL) for 1 hour at 4°C. After that, extender 2 (extender 1 containing 1 M glycerol) was added (v:v) to the diluted sperm, loaded in 0.5-mL straws (1 × 108 sperm/mL), and incubated at 4°C for 30 minutes. The sperm straws were frozen over liquid nitrogen (LN2) vapor for 20 minutes and then plunged directly into LN2. After thawing at 37°C for 25 seconds, sperm progressive motility (CASA), viability (SYBR-14/PI), apoptosis (Annexin V/PI), and reactive oxygen species (ROS; H2DCFDA/PI) were evaluated. Thereafter, the optimal concentrations of trehalose were selected, and the gene expression of BAX, BCL2, NOX5, SMOX, OGG1, and ROMO1 was evaluated after freeze-thawing. Supplementation with 20 and 30 mM trehalose significantly increased sperm progressive motility and viability compared to the control. However, trehalose had no significant effect on sperm ROS or phosphatidylserine translocation index. There were minor numerical increases and decreases in gene expression when the selected optimal concentrations of trehalose (20 and 30 mM) were compared to the control. However, there were no significant differences. We conclude that the addition of trehalose (20 and 30 mM) in EY-free extender could improve sperm motility and viability without significant effects on ROS, apoptosis, or gene expression.

Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations.

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6-9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3-5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments.

Exploring the Influence of Growth-Associated Host Genetics on the Initial Gut Microbiota in Horses.

The influences of diet and environmental factors on gut microbial profiles have been widely acknowledged; however, the specific roles of host genetics remain uncertain. To unravel host genetic effects, we raised 47 Jeju crossbred (Jeju × Thoroughbred) foals that exhibited higher genetic diversity. Foals were raised under identical environmental conditions and diets. Microbial composition revealed that Firmicutes, Bacteroidetes, and Spirochaetes were the predominant phyla. We identified 31 host-microbiome associations by utilizing 47,668 single nucleotide polymorphisms (SNPs) and 734 taxa with quantitative trait locus (QTL) information related to horse growth. The taxa involved in 31 host-microbiome associations were functionally linked to carbohydrate metabolism, energy metabolic processes, short-chain fatty acid (SCFA) production, and lactic acid production. Abundances of these taxa were affected by specific SNP genotypes. Most growth-associated SNPs are found between genes. The rs69057439 and rs69127732 SNPs are located within the introns of the VWA8 and MFSD6 genes, respectively. These genes are known to affect energy balance and metabolism. These discoveries emphasize the significant effect of host SNPs on the development of the intestinal microbiome during the initial phases of life and provide insights into the influence of gut microbial composition on horse growth.

Tackling the Catch-22 Situation of Optimizing a Sensor and a Transporter System in a Whole-Cell Microbial Biosensor Design for an Anthropogenic Small Molecule.

Whole-cell biosensors provide a convenient detection tool for the high-throughput screening of genetically engineered biocatalytic activity. But establishing a biosensor for an anthropogenic molecule requires both a custom transporter and a transcription factor. This results in an unavoidable "Catch-22" situation in which transporter activity cannot be easily confirmed without a biosensor and a biosensor cannot be established without a functional transporter in a host organism. We overcame this type of circular problem while developing an adipic acid (ADA) sensor. First, leveraging an established cis,cis-muconic acid (ccMA) sensor, an annotated ccMA transporter MucK, which is expected to be broadly responsive to dicarboxylates, was stably expressed in the genome of Pseudomonas putida to function as a transporter for ADA, and then a PcaR transcription factor (endogenous to the strain and naturally induced by ß-ketoadipic acid, BKA) was diversified and selected to detect the ADA molecule. While MucK expression is otherwise very unstable in P. putida under strong promoter expression, our optimized mucK expression was functional for over 70 generations without loss of function, and we selected an ADA sensor that showed a specificity switch of over 35-fold from BKA at low concentrations (typically <0.1 mM of inducers). Our ADA and BKA biosensors show high sensitivity (low detection concentration <10 µM) and dynamic range (∼50-fold) in an industrially relevant organism and will open new avenues for high throughput discovery and optimization of enzymes and metabolic pathways for the biomanufacture of these molecules. In particular, the novel ADA sensor will aid the discovery and evolution of efficient biocatalysts for the biological recycling of ADA from the degradation of nylon-6,6 waste.

COVID-19 Vaccine Hesitancy Among Health Care Workers in Four Health Care Systems in Atlanta.

Coronavirus disease 2019 (COVID-19) vaccine hesitancy among health care workers (HCWs) undermines community vaccine confidence. Predictors and reasons for HCW hesitancy in the Atlanta region were evaluated using a survey between May and June 2021. Vaccine hesitancy was highest in younger and less educated HCWs. Interventions to address vaccine hesitancy in HCWs are necessary.

Clinical characteristics and social determinants of health associated with 30-day hospital readmissions of patients with COVID-19.

COVID-19 readmissions are associated with increased patient mortality and healthcare system strain. This retrospective cohort study of PCR-confirmed COVID-19 positive adults (>18 years) hospitalized and readmitted within 30 days of discharge from index admission was performed at eight Atlanta hospitals from March to December 2020. The objective was to describe COVID-19 patient-level demographics and clinical characteristics, and community-level social determinants of health (SDoH) that contribute to 30-day readmissions. Demographics, comorbidities, COVID-19 treatment, and discharge disposition data were extracted from the index admission. ZIP codes were linked to a demographic/lifestyle database interpolating to community-level SDoH. Of 7155 patients with COVID-19, 463 (6.5%) had 30-day, unplanned, all-cause hospital readmissions. Statistically significant differences were not found in readmissions stratified by age, sex, race, or ethnicity. Patients with a high-risk Charlson Comorbidity Index had higher odds of readmission (OR 4.8 (95% CI: 2.1 to 11.0)). Remdesivir treatment and intensive care unit (ICU) care were associated with lower odds of readmission (OR 0.5 (95% CI: 0.4 to 0.8) and OR 0.5 (95% CI: 0.4 to 0.7), respectively). Patients residing in communities with larger average household size were less likely to be readmitted (OR 0.7 (95% CI: 0.5 to 0.9). In this cohort, patients who received remdesivir, were cared for in an ICU, and resided in ZIP codes with higher proportions of residents with increased social support had lower odds of readmission. These patient-level factors and community-level SDoH may be used to identify patients with COVID-19 who are at increased risk of readmission.

Engineered pH-Sensitive Protein G/IgG Interaction.

While natural protein-protein interactions have evolved to be induced by complex stimuli, rational design of interactions that can be switched-on-demand still remain challenging in the protein design world. Here, we demonstrate that a computationally redesigned natural interface for improved binding affinity could further be mutated to adopt a pH switchable interaction. The redesigned interface of Protein G/human IgG Fc domain (referred to as PrG/hIgG), when incorporated with histidine and glutamic acid on PrG (PrG-EHHE), showed a switch in binding affinity by 50-fold when the pH was altered from mild acidic to mild basic. The wild-type (WT) interface showed a negligible switch. The overall binding affinity under mild acidic pH for PrG-EHHE outperformed the wild-type PrG (PrG-WT) interaction. The new reagent PrG-EHHE can be revolutionary in IgG purification, since the standard method of using an extreme acidic pH for elution can be circumvented.

Clinical, behavioural and social factors associated with racial disparities in COVID-19 patients from an integrated healthcare system in Georgia: a retrospective cohort study.

OBJECTIVES: To identify sociodemographic, clinical and behavioural drivers of racial disparities and their association with clinical outcomes among Kaiser Permanente Georgia (KPGA) members with COVID-19. DESIGN: Retrospective cohort of patients with COVID-19 seen from 3 March to 29 October 2020. We described the distribution of underlying comorbidities, quality of care metrics, demographic and social determinants of health (SDOH) indicators across race groups. We also described clinical outcomes in hospitalised patients including length of stay, intensive care unit (ICU) admission, readmission and mortality. We performed multivariable analyses for hospitalisation risk among all patients with COVID-19 and stratifyied by race and sex. SETTING: KPGA, an integrated healthcare system. PARTICIPANTS: 5712 patients who all had laboratory-confirmed COVID-19. Of them, 57.8% were female, 58.4% black, 29.5% white, 8.5% Hispanic and 3.6% Asian. RESULTS: Black patients had the highest proportions of living in neighborhoods under the federal poverty line (12.4%) and in more deprived locations (neighbourhood deprivation index=0.4). Overall, 14.4% (n=827) of this cohort was hospitalised. Asian patients had the highest rates of ICU admission (53.1%) and mechanical ventilation (21.9%). Among all patients, Hispanics (adjusted 1.60, 95% CI (1.08, 2.37)), blacks (1.43 (1.13, 1.83)), age in years (1.03 (1.02, 1.04)) and living in a zip code with high unemployment (1.08 (1.03, 1.13)) were associated with higher odds of hospitalisation. COVID-19 patients with chronic obstructive pulmonary disease (2.59 (1.67, 4.02)), chronic heart failure (1.79 (1.31, 2.45)), immunocompromised (1.77 (1.16, 2.70)), with glycated haemoglobin >8% (1.68 (1.19, 2.38)), depression (1.60 (1.24, 2.06)), hypertension (1.5 (1.21, 1.87)) and physical inactivity (1.25 (1.03, 1.51)) had higher odds of hospitalisation. CONCLUSIONS: Black and Hispanic KPGA patients were at higher odds of hospitalisation, but not mortality, compared with other race groups. Beyond previously reported sociodemographics and comorbidities, factors such as quality of care, lifestyle behaviours and SDOH indicators should be considered when designing and implementing interventions to reduce COVID-19 racial disparities.

Fabrication of TiB2-Al1050 Composites with Improved Microstructural and Mechanical Properties by a Liquid Pressing Infiltration Process.

This study was conducted on titanium diboride (TiB2) reinforced Al metal matrix composites (MMCs) with improved properties using a TiB2 and aluminum (Al) 1050 alloy. Al composites reinforced with fine TiB2 at volume ratios of more than 60% were successfully fabricated via the liquid pressing infiltration (LPI) process, which can be used to apply gas pressure at a high temperature. The microstructure of the TiB2-Al composite fabricated at 1000 °C with pressurization of 10 bar for 1 h showed that molten Al effectively infiltrated into the high volume-fraction TiB2 preform due to the improved wettability and external gas pressurization. In addition, the interface of TiB2 and Al not only had no cracks or pores but also had no brittle intermetallic compounds. In conclusion, TiB2-Al composite, which has a sound microstructure without defects, has improved mechanical properties, such as hardness and strength, due to effective load transfer from the Al matrix to the fine TiB2 reinforcement.

Prediction on Compressive and Split Tensile Strengths of GGBFS/FA Based GPC.

Based on rate constant concept, empirical models were presented for the predictions of age-dependent development of compressive and split tensile strengths of geopolymer concrete composite (GPCC) with fly ash (FA) blended with ground granulated blast furnace slag (GGBFS). The models were empirically developed based on a total of 180 cylindrical test results of GPCC. Six different independent factors comprising of curing temperature, the weight ratios of GGBFS/binder, the aggregate/binder, the alkali solution/binder, the Na2SiO3/NaOH, and the NaOH concentration were considered as the variables. The ANOVA analyses performed on Taguchi orthogonal arrays with six factors in three levels showed that the curing temperature and ratio of GGBFS to binder were the main contributing factors to the development of compressive strength. The models, functionalized with these contributing factors and equivalent age, reflect the level of activation energy of GPCC similar to that of ordinary Portland cement concrete (OPC) and a higher frequency of molecular collisions during the curing period at elevated temperature. The model predictions for compressive and split tensile strength showed good agreements with tested results.

Microstructural Evolution and Strengthening Mechanism of SiC/Al Composites Fabricated by a Liquid-Pressing Process and Heat Treatment.

Aluminum alloy (Al7075) composites reinforced with a high volume fraction of silicon carbide (SiC) were produced by a liquid-pressing process. The characterization of their microstructure showed that SiC particles corresponding to a volume fraction greater than 60% were uniformly distributed in the composite, and Mg2Si precipitates were present at the interface between the matrix and the reinforcement. A superior compressive strength (1130 MPa) was obtained by an effective load transfer to the hard ceramic particles. After solution heat treatment and artificial aging, the Mg2Si precipitates decomposed from rod-shaped large particles to smaller spherical particles, which led to an increase of the compressive strength by more than 200 MPa. The strengthening mechanism is discussed on the basis of the observed microstructural evolution.

Antioxidant and skin-whitening effects of aerial part of Euphorbia supina Raf. Extract.

BACKGROUND: Euphorbia supina (ES) has been widely used in folk medicine owing to its antibacterial, hemostatic, and anti-inflammatory properties. The aim of this study was to evaluate the antioxidant and skin-whitening effects of a 70% ethanol extract of ES. METHODS: The aerial parts of ES plant were extracted with 70% ethanol. The viability of B16F10 cells was evaluated by MTT assay to determine the non-toxic doses for further experiments. The tyrosinase and cellular tyrosinase activities were then measured using an enzyme-substrate assay. In addition, the expression of whitening-related proteins was measured using western blot. RESULTS: The antioxidant activity of the ES samples increased in a dose-dependent manner, as confirmed by their radical scavenging activities in the 2,2-diphenyl-1-1-picrylhydrazyl and 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) assays. The ES extract significantly reduced tyrosinase activity and melanin content in a dose-dependent manner. Furthermore, it decreased α-melanocyte stimulating hormone (MSH)-induced protein expression of tyrosinase and microphthalmia-associated transcription factor (MITF). CONCLUSIONS: Our results indicate that the ES extract attenuated α-MSH-stimulated melanin synthesis by modulating tyrosinase and MITF expression. Therefore, the ES extract could be a promising therapeutic agent to treat hyperpigmentation and as an ingredient for skin-whitening cosmetics.

Methylglyoxal synthase regulates cell elongation via alterations of cellular methylglyoxal and spermidine content in Bacillus subtilis.

Methylglyoxal regulates cell division and differentiation through its interaction with polyamines. Loss of their biosynthesizing enzyme causes physiological impairment and cell elongation in eukaryotes. However, the reciprocal effects of methylglyoxal and polyamine production and its regulatory metabolic switches on morphological changes in prokaryotes have not been addressed. Here, Bacillus subtilis methylglyoxal synthase (mgsA) and polyamine biosynthesizing genes encoding arginine decarboxylase (SpeA), agmatinase (SpeB), and spermidine synthase (SpeE), were disrupted or overexpressed. Treatment of 0.2mM methylglyoxal and 1mM spermidine led to the elongation and shortening of B. subtilis wild-type cells to 12.38±3.21µm (P<0.05) and 3.24±0.73µm (P<0.01), respectively, compared to untreated cells (5.72±0.68µm). mgsA-deficient (mgsA-) and -overexpressing (mgsAOE) mutants also demonstrated cell shortening and elongation, similar to speB- and speE-deficient (speB- and speE-) and -overexpressing (speBOE and speEOE) mutants. Importantly, both mgsA-depleted speBOE and speEOE mutants (speBOE/mgsA- and speEOE/mgsA-) were drastically shortened to 24.5% and 23.8% of parental speBOE and speEOE mutants, respectively. These phenotypes were associated with reciprocal alterations of mgsA and polyamine transcripts governed by the contents of methylglyoxal and spermidine, which are involved in enzymatic or genetic metabolite-control mechanisms. Additionally, biophysically detected methylglyoxal-spermidine Schiff bases did not affect morphogenesis. Taken together, the findings indicate that methylglyoxal triggers cell elongation. Furthermore, cells with methylglyoxal accumulation commonly exhibit an elongated rod-shaped morphology through upregulation of mgsA, polyamine genes, and the global regulator spx, as well as repression of the cell division and shape regulator, FtsZ.

Objective quantification of trochlear dysplasia: Assessment of the difference in morphology between control and chronic patellofemoral instability patients.

BACKGROUND: Trochlear dysplasia is an important risk factor associated with patellofemoral instability, but it remains difficult to classify with consistency. Currently there is no objective way to quantify the dysplasia. The purpose of this study is to define and quantify objectively the trochlear morphology by volume and length via computed tomography (CT). METHODS: One-hundred control patients (136 knees) were retrospectively reviewed and compared to 36 consecutive patients (72 knees) who were treated surgically for recurrent patellar instability and known trochlear dysplasia based on a lateral radiograph. Trochlear morphology was analyzed from a pre-operative CT and data presented as trochlear sulcus volume trochlear length. To determine where along the trochlear length dysplasia is most variable, the trochlear length was radiographically divided into thirds, volume was quantified along that section and compared to control trochlear. RESULTS: A significant difference in trochlear morphology exists between cohorts, volume (1.98 vs 3.77cm3) and length (31.97 vs 34.66mm) (p<0.05). However, there appears to be a gender based difference in trochlea morphology. The trochlea volumetric analysis between the female cohorts (L: 2.02cm3 vs. 2.94cm3, R: 1.95cm3 vs. 2.93cm3) demonstrated significantly less volume in instability patients (p<0.001). The proximal third of the trochlear contributed the majority of dysplasia difference determined by comparing mean trochlear volume, 95% of the difference. This difference decreased in distal sections, 53% and 32% respectively. CONCLUSION: This reproducible technique can be used to quantify the trochlea morphology, in order to describe the severity of a dysplasia.

Genome scan linkage analysis identifies a major quantitative trait loci for fatty acid composition in longissimus dorsi muscle in an F2 intercross between Landrace and Korean native pigs.

OBJECTIVE: This study was conducted to locate quantitative trait loci (QTL) influencing fatty acid (FA) composition in a large F2 intercross between Landrace and Korean native pigs. METHODS: Eighteen FA composition traits were measured in more than 960 F2 progeny. All experimental animals were genotyped with 165 microsatellite markers located throughout the pig autosomes. RESULTS: We detected 112 QTLs for the FA composition; Forty seven QTLs reached the genome-wide significant threshold. In particular, we identified a cluster of highly significant QTLs for FA composition on SSC12. QTL for polyunsaturated fatty acid on pig chromosome 12 (F-value = 97.2 under additive and dominance model, nominal p-value 3.6×10-39) accounted for 16.9% of phenotypic variance. In addition, four more QTLs for C18:1, C18:2, C20:4, and monounsaturated fatty acids on the similar position explained more than 10% of phenotypic variance. CONCLUSION: Our findings of a major QTL for FA composition presented here could provide helpful information to locate causative variants to improve meat quality traits in pigs.

Donor Site Morbidity After Anterior Iliac Bone Graft Harvesting.

OBJECTIVES: The objective of this study was to evaluate donor site morbidity associated with anterior iliac crest bone graft harvesting for orthopaedic trauma reconstructions. DESIGN: Retrospective case series. SETTING: Level 1 trauma center. PATIENTS: Forty-Six consecutive patients who had undergone anterior iliac crest bone graft harvesting more than a 7-year period were reviewed. INTERVENTION: Grafts were taken by removing the inner table of the ilium from 2 cm posterior to the Anterior Superior Iliac Spine. The abductor insertion was left intact and the external contour of the ilium remained unaffected. MAIN OUTCOME MEASURE: Visual analog scale and modified Questionnaires. RESULTS: Thirty-seven of the 46 patients were evaluated at average follow-up of 4.5 years after anterior iliac bone graft harvesting for nonunion or fusion for posttraumatic arthrosis. No patient complained of pain at final follow-up with all patients having a visual analog scale pain of 0 at final follow-up. Three patients reported some scar numbness, however, none complained of thigh numbness. Three patients, if they needed grafting in the future would seek alternative sources based on their remembrance of significant postoperative pain. CONCLUSIONS: Anterior inner table bone graft harvesting resulted in minimal morbidity and neither pain nor functional limitations at an average of 4.5 years. This technique has lower donor-site morbidity than previous reports of posterior graft sites. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Polyamines regulate cell growth and cellular methylglyoxal in high-glucose medium independently of intracellular glutathione.

Polyamines can presumably inhibit protein glycation, when associated with the methylglyoxal inevitably produced during glycolysis. Herein, we hypothesized a nonenzymatic interaction between putrescine and methylglyoxal in putrescine-deficient or -overexpressing Dictyostelium cells in high-glucose medium, which can control methylglyoxal production. Putrescine was essentially required for growth rescue accompanying methylglyoxal detoxification when cells underwent growth defect and cell cycle G1-arrest when supplemented with high glucose. Furthermore, methylglyoxal regulation by putrescine seemed to be a parallel pathway independent of the changes in cellular glutathione content in high-glucose medium. Consequently, we suggest that Dictyostelium cells need polyamines for normal growth and cellular methylglyoxal regulation.

MATLAB/Simulink Pulse-Echo Ultrasound System Simulator Based on Experimentally Validated Models.

A flexible clinical ultrasound system must operate with different transducers, which have characteristic impulse responses and widely varying impedances. The impulse response determines the shape of the high-voltage pulse that is transmitted and the specifications of the front-end electronics that receive the echo; the impedance determines the specification of the matching network through which the transducer is connected. System-level optimization of these subsystems requires accurate modeling of pulse-echo (two-way) response, which in turn demands a unified simulation of the ultrasonics and electronics. In this paper, this is realized by combining MATLAB/Simulink models of the high-voltage transmitter, the transmission interface, the acoustic subsystem which includes wave propagation and reflection, the receiving interface, and the front-end receiver. To demonstrate the effectiveness of our simulator, the models are experimentally validated by comparing the simulation results with the measured data from a commercial ultrasound system. This simulator could be used to quickly provide system-level feedback for an optimized tuning of electronic design parameters.

Intra-articular Fluid Distension for Initial Portal Placement During Hip Arthroscopy: The "Femoral Head Drop" Technique.

Iatrogenic injury is a known complication of initial portal placement during hip arthroscopy. The rate of labral puncture or damage to the articular surfaces with arthroscopic instruments is variable and may be associated with operator inexperience or complex anatomy. In addition, the amount of traction applied to achieve joint distraction may unnecessarily place patients at risk of neurapraxia. The purpose of this article is to describe the "femoral head drop" technique as a method to increase safe access to the central compartment and minimize the amount of traction needed to do so, especially in patients with challenging bony anatomy. This technique uses the application of intra-articular saline solution to cause inferior migration of the femoral head. Intra-articular fluid distension, or the femoral head drop technique, is simple, safe, and reproducible, making it appropriate for hip arthroscopists at any level of experience.

IGF-I improves mitochondrial membrane potential during hypothermic storage of canine spermatozoa.

The aim of study was to evaluate the effects of insulin-like growth factor I (IGF-I) on canine sperm function during cooled and freeze-thaw storage. Extenders supplemented with different IGF-I concentrations (0, 100 and 200 ng/ml) were added to canine spermatozoa, and the sperm samples were stored at 4°C for 48 hr or freeze-thawed. Sperm motility, morphology, plasma-membrane integrity (PMI) and mitochondrial membrane potential (MMP) were evaluated. IGF-I had no effect on PMI or morphology during cooling and freeze-thawing. However, IGF-I alleviated the reduction in progressive motility and MMP caused by cooled storage and led to an improvement in MMP after freeze-thawing. In conclusion, IGF-I can be helpful to maintain progressive motility of canine spermatozoa during hypothermic storage via increased MMP.