Transmission of reduced levels of miR-34/449 from sperm to preimplantation embryos is a key step in the transgenerational epigenetic inheritance of the effects of paternal chronic social instability stress.

The transgenerational effects of exposing male mice to chronic social instability (CSI) stress are associated with decreased sperm levels of multiple members of the miR-34/449 family that persist after their mating through preimplantation embryo (PIE) development. Here we demonstrate the importance of these miRNA changes by showing that restoring miR-34c levels in PIEs derived from CSI stressed males prevents elevated anxiety and defective sociability normally found specifically in their adult female offspring. It also restores, at least partially, levels of sperm miR-34/449 normally reduced in their male offspring who transmit these sex-specific traits to their offspring. Strikingly, these experiments also revealed that inducing miR-34c levels in PIEs enhances the expression of its own gene and that of miR-449 in these cells. The same induction of embryo miR-34/449 gene expression likely occurs after sperm-derived miR-34c is introduced into oocytes upon fertilization. Thus, suppression of this miRNA amplification system when sperm miR-34c levels are reduced in CSI stressed mice can explain how a comparable fold-suppression of miR-34/449 levels can be found in PIEs derived from them, despite sperm containing ~50-fold lower levels of these miRNAs than those already present in PIEs. We previously found that men exposed to early life trauma also display reduced sperm levels of miR-34/449. And here we show that miR-34c can also increase the expression of its own gene, and that of miR-449 in human embryonic stem cells, suggesting that human PIEs derived from men with low sperm miR-34/449 levels may also contain this potentially harmful defect.

Preimplantation Embryos Amplify Stress-Induced Changes in Sperm-Derived miRNA Content to Mediate Transgenerational Epigenetic Inheritance.

Chronically stressing male mice can alter the behavior of their offspring across generations. This effect is thought to be mediated by stress-induced changes in the content of specific sperm miRNAs that modify embryo development after their delivery to oocytes at fertilization. A major problem with this hypothesis is that the levels of mouse sperm miRNAs are much lower than those present in preimplantation embryos. This makes it unclear how embryos could be significantly impacted without an amplification system to magnify changes in sperm miRNA content, like those present in lower organisms where transgenerational epigenetic inheritance is well established. Here, we describe such a system for Chronic Social Instability (CSI) stress that can explain how it reduces the levels of the miR-34b,c/449a,b family of miRNAs not only in sperm of exposed males but also in preimplantation embryos ( PIEs ) derived from their mating, as well as in sperm of male offspring. Sperm-derived miR-34c normally positively regulates expression of its own gene and that of miR-449 in PIEs. This feed forward, auto-amplification process is suppressed when CSI stress reduces sperm miR-34c levels. Its suppression is important for the transmission of traits to offspring because restoring miR-34c levels in PIEs from CSI stressed males, which also restores levels of miR-449 in them, suppresses elements of elevated anxiety and defective sociability normally found specifically in their female offspring, as well as reduced sperm miR-34 and miR-449 levels normally found in male offspring, who pass on these traits to their offspring. We previously published that the content of sperm miR-34/449 is also reduced in men raised in highly abusive and/or dysfunctional families. We show here that a similar miRNA auto-amplification system functions in human embryonic stem cells. This raises the possibility that PIEs in offspring of these men also display reduced levels of miR-34/449, enhancing the potential translational significance of these studies.

Validation of a focused echocardiographic training program in first opinion practice.

BACKGROUND: The EPIC study defines criteria, including echocardiographic assessments of left atrial to aortic ratio (LA: Ao) and left ventricular internal diameter in diastole normalized for body weight (LVIDdN), for dogs with preclinical myxomatous mitral valve disease (MMVD) likely to benefit from pimobendan therapy. Access to echocardiography by a cardiologist is not universally available. HYPOTHESIS/OBJECTIVES: Completion of a focused echocardiographic training program would result in accurate identification of dogs fulfilling the EPIC criteria by primary care veterinarians (PC). PARTICIPANTS: Six PCs with no previous echocardiographic experience. METHODS: Prospective diagnostic test accuracy study. After training, each PC evaluated ≤10 dogs that they believed to have preclinical MMVD. The evaluation was repeated by 1 of 3 cardiology diplomates, blinded to the PC's findings. Agreement between clinical assessments and echocardiographic measurements was assessed. RESULTS: Fifty-seven dogs were evaluated by PCs; 1 dog was withdrawn because of congestive heart failure. The median time between PC and cardiologist evaluation was 0 days (range, 0-8). One incorrect diagnosis of MMVD was made by a PC (this dog had dilated cardiomyopathy [DCM]); preclinical MMVD was confirmed by the cardiologist in 55 dogs. No difference in LA: Ao (P = .96; CV = 6.9%) was detected between PCs and cardiologists. LVIDdN (1.69 cm/kg0.294 (1.26-2.21) vs 1.73 cm/kg0.294 (1.32-2.73); P = .001; CV = 6.5%), was significantly lower when measured by PCs vs cardiologists. PCs and cardiologists agreed regarding assessment of EPIC criteria in 49/56 dogs (Alpha = .761, 95% confidence interval 0.697-0.922). CONCLUSIONS AND CLINICAL IMPORTANCE: The program effectively trained PCs to accurately assess EPIC criteria in dogs with preclinical MMVD.

nsink: An R package for flow path nitrogen removal estimation.

The nsink package estimates cumulative nitrogen (N) removal along a specified flow path and is based on methodologies outlined in Kellogg et al. (2010). For a user-specified watershed (i.e., hydrologic unit code (HUC)), nsink downloads all required datasets from public datasets in the United States, prepares data for use, summarizes N removal along a flow path and creates several static maps. The results of an nsink analysis may be exported to standard geospatial files for use in other applications.

The 2nd Metacarpal Cortical Index as a Simple Screening Tool for Osteopenia.

BACKGROUND: Osteopenia is a known risk factor for sustaining skeletal fractures. Prevention of fragility fractures has obvious clinical and economic advantages, however screening all patients using a dual energy X-ray absorptiometry (DXA) is controversial not only because of the cost implications but also because it would potentially involve exposing a percentage of normal patients to unnecessary radiation. We wished to assess whether a simple hand X-ray measuring the 2nd metacarpal cortical index (2MCI) could be used as a simple screening tool for identifying patients with osteopenia. METHODS: We retrospectively collected the radiographic data of 206 patients who had a simple radiograph of the hand and a DXA scan within one year of each other from our picture archiving and communication system database. The 2MCI was calculated for all patients. As data was parametric, a Pearson's correlation was performed to assess association between Tscores and 2MCI. Further analysis involved the construction of receiver operating characteristic (ROC) curves to identify a 2MCI index, which would give the most appropriate sensitivity and specificity values for identifying the presence of osteopenia. RESULTS: A statistically significant and moderate correlation between DXA T-scores and 2MCI values was found (r=0.54, n=206, P<0.001). Further ROC curve analysis of normal and osteopenic subjects identified that a 2MCI of 41.5 had a sensitivity of 100% and specificity of 53% for detecting osteopenia. CONCLUSIONS: Our results support the use of the 2MCI as a simple screening tool for identifying patients with osteopenia.

Outcomes and Early Recurrence Following Enzymatic (Collagenase) Treatment of Moderate and Severe Dupuytren Contractures.

PURPOSE: We offer collagenase Clostridium histolyticum (CCH) injections to all patients with a Dupuytren contracture and a palpable cord. We assessed whether more severe contractures respond less well or recur more frequently. METHODS: From a database of 502 CCH injections, 386 (77%) had a complete dataset with minimum 1-year face-to-face follow-up. Contracture severity was assessed using the Tubiana system: grade 1 (27%), grade 2 (49%), grade 3 (19%), and grade 4 (5%). Patients received a single intralesional injection of 0.58 mg CCH followed by manipulation. Finger position was measured at 6 to 12 weeks, 6 months, and 1 year. Failure to break the cord, skin tears, and any adverse events were noted. Recurrence was defined as a failure to maintain any prior correction to within 20°. RESULTS: There were 17 failures (4%) and 6 allergic reactions (1%). We found 31% corrected completely, with approximately half remaining corrected at 1 year. We achieved 43° (95% confidence interval, 40°-46°) correction of combined deformity, with 11° (95% confidence interval, 9°-13°) correction attrition over 1 year. Sixteen percent of treated digits experienced a recurrence with no difference between Tubiana grades, and one-third chose further treatment. Following treatment, Tubiana grade 1 contractures improved by 78% in comparison with higher grade contractures (55%-67% relative correction). By 1 year, all grades had a similar mean 46% relative correction. A complete correction was seen in 61% of grade 1 contractures, with more severe contractures less likely to correct completely (9%-24%). A 31% skin tear rate had no impact on outcome, recurrence, or long-term morbidity. CONCLUSIONS: CCH may treat any Dupuytren cord regardless of severity. Although contracture may recur, few patients pursue further treatment within 1 year. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Involvement of early embryonic miR-409-3p in the establishment of anxiety levels in female mice.

Small RNA molecules in early embryos, delivered from sperm to zygotes upon fertilization, are required for normal mouse embryonic development. Even modest changes in the levels of sperm-derived miRNAs appear to influence early embryos and subsequent development. For example, stress-associated behaviors develop in mice after injection into normal zygotes sets of sperm miRNAs elevated in stressed male mice. Here, we implicate early embryonic miR-409-3p in establishing anxiety levels in adult female, but not male mice. First, we found that exposure of male mice to chronic social instability stress, which leads to elevated anxiety in their female offspring across at least three generations through the paternal lineage, elevates sperm miR-409-3p levels not only in exposed males, but also in sperm of their F1 and F2 male offspring. Second, we observed that while injection of a mimic of miR-409-3p into zygotes from mating control males was incapable of mimicking this effect in offspring derived from them, injection of a specific inhibitor of this miRNA led to the opposite, anxiolytic effect in female, but not male, and offspring. These findings imply that baseline miR-409-3p activity in early female embryos is necessary for the expression of normal anxiety levels when they develop into adult females. In addition, elevated embryo miR-409-3p activity, possibly as a consequence of stress-induced elevation of its expression in sperm, may participate in, but may not be sufficient for, the induction of enhanced anxiety.

RASGRF1 in CRF cells controls the early adolescent female response to repeated stress.

RASGRF1 (GRF1) is a calcium-stimulated guanine-nucleotide exchange factor that activates RAS and RAC GTPases. In hippocampus neurons, it mediates the action of NMDA and calcium-permeable AMPA glutamate receptors on specific forms of synaptic plasticity, learning, and memory in both male and female mice. Recently, we showed GRF1 also regulates the HPA axis response to restraint stress, but only in female mice before puberty. In particular, we found that after 7 days of restraint stress (7DRS) (30 min/day) both elevated serum CORT levels and induction of an anxiolytic phenotype normally observed in early adolescent (EA) female mice are blocked in GRF1-knockout mice. In contrast, no effects were observed in EA male or adult females. Here, we show this phenotype is due, at least in part, to GRF1 loss in CRF cells of the paraventricular nucleus of the hypothalamus, as GRF1 knockout specifically in these cells suppressed 7DRS-induced elevation of serum CORT levels specifically in EA females, but only down to levels found in comparably stressed EA males. Nevertheless, it still completely blocked the 7DRS-induced anxiolytic phenotype observed in EA females. Interestingly, loss of GRF1 in CRF cells had no effect after only three restraint stress exposures, implying a role for GRF1 in 7DRS stress-induced plasticity of CRF cells that appears to be specific to EA female mice. Overall, these findings indicate that GRF1 in CRF cells makes a key contribution to the distinct response EA females display to repeated stress.

The 27 kDa Trypanosoma brucei Pentatricopeptide Repeat Protein is a G-tract Specific RNA Binding Protein.

Pentatricopeptide repeat (PPR) proteins, a helical repeat family of organellar RNA binding proteins, play essential roles in post-transcriptional RNA processing. In Trypanosoma brucei, an expanded family of PPR proteins localize to the parasite's single mitochondrion, where they are believed to perform important roles in both RNA processing and translation. We studied the RNA binding specificity of the simplest T. brucei PPR protein (KRIPP11) using electrophoretic mobility shift assays, fluorescence anisotropy, circular dichroism spectroscopy, and in vitro selection. We found KRIPP11 to be an RNA binding protein with specificity for sequences of four or more consecutive guanosine residues (G-tracts). Such G-tracts are dramatically enriched in T. brucei mitochondrial transcripts that are destined for extensive uridine insertion/deletion editing but are not present in mRNAs following editing. We further found that the quadruplex oligoguanosine RNA conformation is preferentially recognized by KRIPP11 over other conformational forms, and is bound without disruption of the quadruplex structure. In combination with prior data demonstrating association of KRIPP11 with the small ribosomal subunit, these results suggest possible roles for KRIPP11 in bridging mRNA maturation and translation or in facilitating translation of unusual dual-coded open reading frames.

Reduced levels of miRNAs 449 and 34 in sperm of mice and men exposed to early life stress.

Exposure of male mice to early life stress alters the levels of specific sperm miRNAs that promote stress-associated behaviors in their offspring. To begin to evaluate whether similar phenomena occur in men, we searched for sperm miRNA changes that occur in both mice and men exposed to early life stressors that have long-lasting effects. For men, we used the Adverse Childhood Experience (ACE) questionnaire. It reveals the degree of abusive and/or dysfunctional family experiences when young, which increases risks of developing future psychological and physical disorders. For male mice, we used adolescent chronic social instability (CSI) stress, which not only enhances sociability defects for >1 year, but also anxiety and defective sociability in female offspring for multiple generations through the male lineage. Here we found a statistically significant inverse correlation between levels of multiple miRNAs of the miR-449/34 family and ACE scores of Caucasian males. Remarkably, we found members of the same sperm miRNA family are also reduced in mice exposed to CSI stress. Thus, future studies should be designed to directly test whether reduced levels of these miRNAs could be used as unbiased indicators of current and/or early life exposure to severe stress. Moreover, after mating stressed male mice, these sperm miRNA reductions persist in both early embryos through at least the morula stage and in sperm of males derived from them, suggesting these miRNA changes contribute to transmission of stress phenotypes across generations. Since offspring of men exposed to early life trauma have elevated risks for psychological disorders, these findings raise the possibility that a portion of this risk may be derived from epigenetic regulation of these sperm miRNAs.

Effect of immobilization of the distal interphalangeal joint of fingers on grip strength.

Arthrodesis of the distal interphalangeal joint of the finger is an effective salvage treatment for end-stage arthropathy of the joint. This study aimed to evaluate the effect of simulated fusion of individual distal interphalangeal joints on the overall grip strength of the hand. Custom moulded thermoplastic splints were used to simulate fusion by immobilizing the index, middle, ring and little fingers' distal interphalangeal joints in turn in both hands of 56 healthy participants. Testing was performed with no immobilization and after immobilization of each of the individual digits. Grip strengths reduced significantly following immobilization of the distal interphalangeal joint. The degree of reduction became progressively more pronounced from the index to the little fingers (12%, 18%, 24% and 25%, respectively) and was similar for the dominant and non-dominant hands. This information may have clinical application when counselling patients regarding fusion of the distal interphalangeal joint of the fingers.

Two-dimensional echocardiographic estimates of left atrial function in healthy dogs and dogs with myxomatous mitral valve disease.

OBJECTIVES: To provide reference intervals for 2-dimensional linear and area-based estimates of left atrial (LA) function in healthy dogs and to evaluate the ability of estimates of LA function to differentiate dogs with subclinical myxomatous mitral valve disease (MMVD) and similarly affected dogs with congestive heart failure (CHF). ANIMALS: Fifty-two healthy adult dogs, 88 dogs with MMVD of varying severity. METHODS: Linear and area measurements from 2-dimensional echocardiographs in both right parasternal long and short axis views optimized for the left atrium were used to derive estimates of LA active emptying fraction, passive emptying fraction, expansion index, and total fractional emptying. Differences for each estimate were compared between healthy and MMVD dogs (based on ACVIM classification), and between MMVD dogs with subclinical disease and CHF that had similar LA dimensions. Diagnostic utility at identifying CHF was examined for dogs with subclinical MMVD and CHF. Relationships with bodyweight were assessed. RESULTS: All estimates of LA function decreased with increasing ACVIM stage of mitral valve disease (p<0.05) and showed negative relationships with increasing LA size (all r2 values < 0.2), except for LA passive emptying fraction, which did not differ or correlate with LA size (p=0.4). However, no index of LA function identified CHF better than measurements of LA size. Total LA fractional emptying and expansion index showed modest negative correlations with bodyweight. CONCLUSIONS: Estimates of LA function worsen with worsening MMVD but fail to discriminate dogs with CHF from those with subclinical MMVD any better than simple estimates of LA size.

Pyrocarbon Proximal Interphalangeal Joint Arthroplasty: Minimum Five-Year Follow-Up.

PURPOSE: To report the outcomes, complications, and survivorship of pyrocarbon proximal interphalangeal joint arthroplasty at a minimum of 5-year follow-up. METHODS: A review of 97 implants in 72 consecutive patients from our joint arthroplasty database was undertaken. Patient demographics, complications, further surgery, and implant revision were recorded. Objective outcome was assessed by grip strength, range of motion, and radiological assessment of alignment, loosening, and subsidence. Subjective outcome was assessed by Patient Evaluation Measure; Quick Disabilities of the Arm, Shoulder, and Hand score; and visual analog scores (0, best; 10, worst) for appearance, satisfaction, and pain. RESULTS: Diagnosis was osteoarthritis in 60 joints, rheumatoid arthritis in 12 joints, psoriatic arthritis in 11 joints, and trauma in 14 joints. The average follow-up was 118 months (range, 60-164 months). The mean arc of motion was 35° (range, 0° to 90°). There was no difference in grip strength between operated and nonoperated side. Of the 97 implants, 36 required additional surgery, of which 14 were revised and 22 required reconstruction around a retained implant. The average Patient Evaluation Measure and Quick Disabilities of the Arm, Shoulder and Hand scores were 33 (range, 10-69) and 35 (range, 0-93), respectively. Mean visual analog scores for pain, satisfaction, and appearance were 2 (range, 0-8), 7 (0-10), and 8 (0-10), respectively. All implants had a lucent line with nearly all classified as either Herren grade 2 or 3. Progressive loosening was seen in 48% of implants. Implant survival as assessed by Kaplan-Meier was 85% at both 5 and 10 years. CONCLUSIONS: Good pain relief and maintenance of preoperative arc of motion was achieved with no major deterioration over time. Most implant revisions were performed within 24 months of the index procedure. Currently progressive loosening was not translated into revision surgery. Implant revision rate was higher than with other prostheses. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Pyrocarbon Metacarpophalangeal Joint Arthroplasty in Noninflammatory Arthritis: Minimum 5-Year Follow-Up.

PURPOSE: To report the outcomes, complications, and survivorship of pyrocarbon metacarpophalangeal joint arthroplasty in noninflammatory arthropathy at a minimum 5-year follow-up. METHODS: A retrospective review of 51 implants in 36 patients was undertaken. Patient demographics, complications, further surgery, and implant revision were recorded. Objective outcome was assessed by grip strength, range of motion, and radiological assessment of alignment, loosening, and subsidence. Subjective outcome was assessed by Patient Evaluation Measure, Quick Disabilities of the Arm, Shoulder and Hand score, and visual analog scores (0, best; 10, worst) for appearance, satisfaction, and pain. RESULTS: There were 35 index and 16 middle fingers. The average follow-up was 103 months (range, 60-172 months). The mean arc of motion was 54° (range, 20° to 80°). There was no difference in grip strength between operated and nonsurgical side. Six implants were revised, and 3 of these required additional surgery. The average Patient Evaluation Measure and Quick Disabilities of the Arm, Shoulder and Hand scores were 27 (range, 10-54) and 29 (range, 0-57), respectively. Mean visual analog scores for pain, satisfaction, and appearance were all 1 with the respective ranges being 0-7, 0-4, and 0-6. Most implants were Herren grade 1 lucency with the remaining 5 proximal and 12 distal implants being grade 2. Mean subsidence in the proximal component was 2 mm (range, 0-4 mm) and 1 mm (range, 0-3 mm) in the distal component. The degree of loosening or subsidence did not correlate with outcome. Implant survival as assessed by Kaplan-Meier was 88% at 10 years. CONCLUSIONS: Good pain relief, a functional range of motion, and high satisfaction were seen in the majority of patients. All implant revisions were performed within 18 months of the index procedure. This may represent technical issues rather than problems with the implant.

Silica sol-gel encapsulation of cyanobacteria: lessons for academic and applied research.

Cyanobacteria inhabit nearly every ecosystem on earth, play a vital role in nutrient cycling, and are useful as model organisms for fundamental research in photosynthesis and carbon and nitrogen fixation. In addition, they are important for several established biotechnologies for producing food additives, nutritional and pharmaceutical compounds, and pigments, as well as emerging biotechnologies for biofuels and other products. Encapsulation of living cyanobacteria into a porous silica gel matrix is a recent approach that may dramatically improve the efficiency of certain production processes by retaining the biomass within the reactor and modifying cellular metabolism in helpful ways. Although encapsulation has been explored empirically in the last two decades for a variety of cell types, many challenges remain to achieving optimal encapsulation of cyanobacteria in silica gel. Recent evidence with Synechocystis sp. PCC 6803, for example, suggests that several unknown or uncharacterized proteins are dramatically upregulated as a result of encapsulation. Also, additives commonly used to ease stresses of encapsulating living cells, such as glycerol, have detrimental impacts on photosynthesis in cyanobacteria. This mini-review is intended to address the current status of research on silica sol-gel encapsulation of cyanobacteria and research areas that may further the development of this approach for biotechnology applications.

Diffusion of dissolved ions from wet silica sol-gel monoliths: implications for biological encapsulation.

Divalent nickel (Ni(2+)), Cu(II)EDTA, methyl orange, and dichromate were used to investigate diffusion from hydrated silica sol-gel monoliths. The objective was to examine diffusion of compounds on a size regime relevant to supporting biological components encapsulated within silica gel prepared in a biologically compatible process space with no post-gelation treatments. With an initial sample set, gels prepared from tetraethoxysilane were explored in a factorial design with Ni(2+) as the tracer, varying water content during hydrolysis, acid catalyst present during hydrolysis, and the final concentration of silica. A second sample set explored diffusion of all four tracers in gels prepared with aqueous silica precursors and a variety of organically modified siloxanes. Excluding six outliers which displayed significant syneresis, the mean diffusion constant (D(gel)) across the entire process space of sample set 1 was 2.42×10(-10) m(2) s(-1); approximately 24% of the diffusion coefficient of Ni(2+) in unconfined aqueous solution. In sample set 2, the tracer size and not gel hydrophobicity was the primary determinant of changes in diffusion rates. A strong linear inverse correlation was found between tracer size and the magnitude of D(gel). Based on correlation with the tracers used in this investigation, the characteristic 1-h diffusion distance for carbonate species relevant to supporting active phototrophic organisms was approximately 1.5mm. These results support the notion that silica sol-gel formulations may be optimized for a given biological entity of interest with manageable impact to the diffusion of small ions and molecules.

Transcriptomic responses of Synechocystis sp. PCC 6803 encapsulated in silica gel.

Global gene expression of Synechocystis sp. PCC 6803 encapsulated in silica gel was examined by microarray analysis. Cultures were encapsulated in gels derived from aqueous precursors or from alkoxide precursors and incubated under constant light for 24 h prior to RNA extraction. Cultures suspended in liquid media were exposed to 500 mM salt stress and incubated under identical conditions for comparison purposes. The expression of 414 genes was significantly altered by encapsulation in aqueous-derived gels (fold change ≥1.5 and P value < 0.01), the expression of 1,143 genes was significantly altered by encapsulation in alkoxide-derived gels, and only 243 genes were common to both encapsulation chemistries. Additional qRT-PCR analyses of four selected genes, ggpS, cpcG2, slr5055, and sll5057, confirmed microarray results for those genes. These results illustrate that encapsulation stress is quite different than salt stress in terms of gene expression response. Furthermore, a number of hypothetical and unknown proteins associated with encapsulation and alcohol stress have been identified with implications for improving encapsulation protocols and rationally engineering microorganisms for direct biofuel production.