Exploring 3D leaf anatomical traits for C4 photosynthesis: chloroplast and plasmodesmata pit field size in maize and sugarcane.

Volume and surface area of chloroplasts and surface area of plasmodesmata pit fields are presented for two C4 species, maize and sugarcane, with respect to cell surface area and cell volume. Serial block face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy with the Airyscan system (LSM) were used. Chloroplast size estimates were much faster and easier using LSM than with SBF-SEM; however, the results were more variable than SBF-SEM. Mesophyll cells were lobed where chloroplasts were located, facilitating cell-to-cell connections while allowing for greater intercellular airspace exposure. Bundle sheath cells were cylindrical with chloroplasts arranged centrifugally. Chloroplasts occupied c. 30-50% of mesophyll cell volume, and 60-70% of bundle sheath cell volume. Roughly 2-3% of each cell surface area was covered by plasmodesmata pit fields for both bundle sheath and mesophyll cells. This work will aid future research to develop SBF-SEM methodologies with the aim to better understand the effect of cell structure on C4 photosynthesis.

Pes Planovalgus Is Associated with Increased Comorbidities and Poor Outcomes After Total Knee Arthroplasty.

Pes planovalgus affects knee biomechanics but there are no studies describing its impact on total knee arthroplasty (TKA). We aim to characterize the demographics, medical, and surgical complications of patients with pes planovalgus undergoing TKA. A Medicare database was queried using ICD-9 codes to identify 5,750 patients with and 23,000 patients without pes planovalgus who underwent TKA from 2005 to 2014. Standard descriptive statistics were used to compare medical and surgical complications at 90 days and 2 years, with alpha < 0.003 after a Bonferroni Correction. Patients with pes planovalgus had an elevated incidence of hypertension (80%, p < 0.001), pulmonary disease (31%, p < 0.001), hypothyroidism (28%, p < 0.001), diabetes (30%, p < 0.001), vascular disease (20%, p < 0.001), obesity (26%, p < 0.001), and depression (23%, p < 0.001). They also had increased odds of deep vein thrombosis (DVT) (odds ratio [OR] 1.3, p < 0.001), stiffness (OR 1.3, p < 0.003) and revision (OR 1.59, p < 0.003) at 90 days. At 2 years, odds of stiffness had increased (OR 1.34, p < 0.001) with similar rates of revision and medical complications. Pes planovaglus is associated with increased medical comorbidities and this patient population may be at an increased risk for postoperative stiffness, early revisions, and DVT after TKA. Arthroplasty surgeons should be conscious of these risks when considering TKA in a patient with pes planovalgus and counsel them appropriately. (Journal of Surgical Orthopaedic Advances 32(3):202-206, 2023).

Cadherin puncta are interdigitated dynamic actin protrusions necessary for stable cadherin adhesion.

Cadherins harness the actin cytoskeleton to build cohesive sheets of cells using paradoxically weak bonds, but the molecular mechanisms are poorly understood. In one popular model, actin organizes cadherins into large, micrometer-sized clusters known as puncta. Myosin is thought to pull on these puncta to generate strong adhesion. Here, however, we show that cadherin puncta are actually interdigitated actin microspikes generated by actin polymerization mediated by three factors (Arp2/3, EVL, and CRMP-1). The convoluted membranes in these regions give the impression of cadherin clustering by fluorescence microscopy, but the ratio of cadherin to membrane is constant. Nevertheless, these interlocking fingers of membrane are important for adhesion because perturbing their formation disrupts cell adhesion. In contrast, blocking myosin-dependent contractility does not disrupt either the interdigitated microspikes or lateral membrane adhesion. "Puncta" are zones of strong cell-cell adhesion not due to cadherin clustering but that occur because the interdigitated microspikes expand the surface area available for adhesive bond formation and increase the asperity of the cell surface to promote friction between cells.

Complementary Oligonucleotide Conjugated Multicolor Carbon Dots for Intracellular Recognition of Biological Events.

By using complementary DNA sequences as surface ligands, we selectively allow two individual diffusing "dual-color" carbon dots to interact in situ and in vitro. Spontaneous nanoscale oxidation of surface-abundant nitroso-/nitro-functionalities leads to two distinctly colored carbon dots (CD) which are isolated by polarity driven chromatographic separation. Green- and red-emitting carbon dots (gCD and rCD) were decorated by complementary single-stranded DNAs which produce a marked increase in the fluorescence emission of the respective carbon dots. Mutual colloidal interactions are achieved through hybridization of complementary DNA base pairs attached to the respective particles, resulting in quenching of their photoluminescence. The observed post-hybridization quenching is presumably due to a combined effect from an aggregation of CDs post duplex DNA formation and close proximity of multicolored CDs, having overlapped spectral regions leading to a nonradiative energy transfer process possibly released as heat. This strategy may contribute to the rational design of mutually interacting carbon dots for a better control over the resulting assembly structure for studying different biological phenomenon including molecular cytogenetics. One of the newly synthesized CDs was successfully used to image intracellular location of GAPDH mRNA using an event of change in fluorescence intensity (FI) of CDs. This selectivity was introduced by conjugating an oligonucleotide harboring complementary sequence to GAPDH mRNA. FI of this conjugated carbon dot, rCD-GAPDH, was also found to decrease in the presence of Ca2+, varied in relation to H+ concentrations, and could serve as a tool to quantify the intracellular concentrations of Ca2+ and pH value (H+) which can give important information about cell survival. Therefore, CD-oligonucleotide conjugates could serve as efficient probes for cellular events and interventions.

Current patterns of genetic diversity in indigenous and introduced species of land snails in Cameroon reflect isolation by distance, limited founder size and known evolutionary relationships.

The aim of this study was to gain molecular insights into patterns of genetic diversity in indigenous and introduced land snails in Cameroon. These species, Archachatina marginata and Achatina fulica, form an important source of protein in Cameroon, but widespread utilization may possibly impact natural patterns of genetic diversity of the indigenous species, while the introduced species may display signs of genetic drift. The evolutionary relationship between the indigenous and introduced species was also studied. Specimens were collected from seven sites in Cameroon. Genetic analyses using COI mitochondrial DNA data suggest that gene flow among the Ar. marginata populations screened follows a model of isolation by distance, and genetic diversity estimates for this species did not provide support for the hypothesis of loss of genetic diversity in areas of intense harvesting. Diversity in the introduced species was much lower, which is likely the signature of an introduction involving limited numbers.

High density genome wide genotyping-by-sequencing and association identifies common and low frequency SNPs, and novel candidate genes influencing cow milk traits.

High-throughput sequencing technologies have increased the ability to detect sequence variations for complex trait improvement. A high throughput genome wide genotyping-by-sequencing (GBS) method was used to generate 515,787 single nucleotide polymorphisms (SNPs), from which 76,355 SNPs with call rates >85% and minor allele frequency ≥1.5% were used in genome wide association study (GWAS) of 44 milk traits in 1,246 Canadian Holstein cows. GWAS was accomplished with a mixed linear model procedure implementing the additive and dominant models. A strong signal within the centromeric region of bovine chromosome 14 was associated with test day fat percentage. Several SNPs were associated with eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, CLA:9c11t and gamma linolenic acid. Most of the significant SNPs for 44 traits studied are novel and located in intergenic regions or introns of genes. Novel potential candidate genes for milk traits or mammary gland functions include ERCC6, TONSL, NPAS2, ACER3, ITGB4, GGT6, ACOX3, MECR, ADAM12, ACHE, LRRC14, FUK, NPRL3, EVL, SLCO3A1, PSMA4, FTO, ADCK5, PP1R16A and TEP1. Our study further demonstrates the utility of the GBS approach for identifying population-specific SNPs for use in improvement of complex dairy traits.

Associations between variants of FADS genes and omega-3 and omega-6 milk fatty acids of Canadian Holstein cows.

BACKGROUND: Fatty acid desaturase 1 (FADS1) and 2 (FADS2) genes code respectively for the enzymes delta-5 and delta-6 desaturases which are rate limiting enzymes in the synthesis of polyunsaturated omega-3 and omega-6 fatty acids (FAs). Omega-3 and-6 FAs as well as conjugated linoleic acid (CLA) are present in bovine milk and have demonstrated positive health effects in humans. Studies in humans have shown significant relationships between genetic variants in FADS1 and 2 genes with plasma and tissue concentrations of omega-3 and-6 FAs. The aim of this study was to evaluate the extent of sequence variations within these two genes in Canadian Holstein cows as well as the association between sequence variants and health promoting FAs in milk. RESULTS: Thirty three SNPs were detected within the studied regions of genes including a synonymous mutation (FADS1-07, rs42187261, 306Tyr > Tyr) in exon 8 of FADS1, a non-synonymous mutation (FADS2-14, rs211580559, 294Ala > Val) within FADS2 exon 7, a splice site SNP (FADS2-05, rs211263660), a 3'UTR SNP (FADS2-23, rs109772589), and another 3'UTR SNP with an effect on a microRNA binding site within FADS2 gene (FADS2-19, rs210169303). Association analyses showed significant relations between three out of seven tested SNPs and several FAs. Significant associations (FDR P < 0.05) were recorded between FADS2-23 (rs109772589) and two omega-6 FAs (dihomogamma linolenic acid [C20:3n6] and arachidonic acid [C20:4n6]), FADS1-07 (rs42187261) and one omega-3 FA (eicosapentaenoic acid, C20:5n3) and tricosanoic acid (C23:0), and one intronic SNP, FADS1-01 (rs136261927) and C20:3n6. CONCLUSION: Our study has demonstrated positive associations between three SNPs within FADS1 and FADS2 genes (a SNP within the 3'UTR, a synonymous SNP and an intronic SNP), with three milk PUFAs of Canadian Holstein cows thus suggesting possible involvement of synonymous and non-coding region variants in FA synthesis. These SNPs may serve as potential genetic markers in breeding programs to increase milk FAs that are of benefit to human health.

Application of the Brixton spatial anticipation test in stroke: ecological validity and performance characteristics.

Executive dysfunction predicts functional recovery post-stroke. However, traditional neuropsychological tests have limitations with this population due to required verbal response, complex motor response, and lengthy administration time. This study examined the ecological validity and performance characteristics of a relatively new measure of executive function, the Brixton Spatial Anticipation Test, which does not place the aforementioned demands on these patients. A total of 57 ischemic stroke patients with frontal lobe and subcortical lesions were administered the Brixton, on average 2 weeks post-stroke, during inpatient rehabilitation and assessed using the Functional Independence Measure (FIM) upon discharge. Brixton performance was significantly correlated with the FIM Total and FIM Cognitive Subtotal at discharge but unrelated to FIM Motor Subtotal. After controlling for global ability measured by the RBANS, Brixton performance accounted for additional variance in predicting FIM Total score at discharge. Interestingly, patients with subcortical strokes in the thalamus and basal ganglia performed significantly worse than patients with strokes in the frontal cortex on the Brixton, supporting the role of subcortical structures in the frontal lobe circuitry and executive function. Based on the present findings, the Brixton is a sensitive measure conducive to the stroke population and has strong ecological validity for identifying cognitive functional outcomes post-stroke.

The dual role of HOP2 in mammalian meiotic homologous recombination.

Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2-MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1(-/-) spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2-MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis.

Dynamics of response to asynapsis and meiotic silencing in spermatocytes from Robertsonian translocation carriers.

Failure of homologous synapsis during meiotic prophase triggers transcriptional repression. Asynapsis of the X and Y chromosomes and their consequent silencing is essential for spermatogenesis. However, asynapsis of portions of autosomes in heterozygous translocation carriers may be detrimental for meiotic progression. In fact, a wide range of phenotypic outcomes from meiotic arrest to normal spermatogenesis have been described and the causes of such a variation remain elusive. To better understand the consequences of asynapsis in male carriers of Robertsonian translocations, we focused on the dynamics of recruitment of markers of asynapsis and meiotic silencing at unsynapsed autosomal trivalents in the spermatocytes of Robertsonian translocation carrier mice. Here we report that the enrichment of breast cancer 1 (BRCA1) and histone γH2AX at unsynapsed trivalents declines during the pachytene stage of meiosis and differs from that observed in the sex body. Furthermore, histone variant H3.3S31, which associates with the sex chromosomes in metaphase I/anaphase I spermatocytes, localizes to autosomes in 12% and 31% of nuclei from carriers of one and three translocations, respectively. These data suggest that the proportion of spermatocytes with markers of meiotic silencing of unsynapsed chromatin (MSUC) at trivalents depends on both, the stage of meiosis and the number of translocations. This may explain some of the variability in phenotypic outcomes associated with Robertsonian translocations. In addition our data suggest that the dynamics of response to asynapsis in Robertsonian translocations differs from the response to sex chromosomal asynapsis in the male germ line.

Inorganic and organozinc fluorocarboxylates: synthesis, structure and materials chemistry.

The organozinc fluorocarboxylates RZnO2CRf and RZnO2CRf·TMEDA, along with Zn(O2CRf)2·TMEDA (R = Me, Et; Rf = C2F5, C3F7) have been synthesized. The structures of EtZnO2C2F5 (5), EtZnO2C3F7 (7), EtZnO2C2F5·TMEDA (11), Zn(O2C2F5)2·TMEDA (13), along with products from the adventitious reaction with either O2 or H2O, Zn10Me4(OMe)4(O2CC2F5)12 (2), Zn9Et2(O2CC2F5)12(O)2 (6), Zn8Et4(OEt)4(O2CC3F7)6(O) (8), [Zn(O2CC3F7)2·TMEDA]2·H2O (15) have been determined. Thin films of oriented ZnO have been deposited on glass substrates by low-pressure chemical vapor deposition (LPCVD) using 3 and 10 as precursors, though no fluorine incorporation in the films was noted. LPCVD using 13 as precursor also yielded fluorine-free ZnO, but lacking the oriented growth observed using 3, 10. However, 5, which exhibits short intermolecular Zn···F contacts in the solid state, thermally decomposes to bulk ZnF2.

Homologous pairing preceding SPO11-mediated double-strand breaks in mice.

How homologous chromosomes (homologs) find their partner, pair, and recombine during meiosis constitutes the central phenomenon in eukaryotic genetics. It is widely believed that, in most organisms, SPO11-mediated DNA double-strand breaks (DSBs) introduced during prophase I precede and are required for efficient homolog pairing. We now show that, in the mouse, a significant level of homolog pairing precedes programmed DNA cleavage. Strikingly, this early chromosome pairing still requires SPO11 but is not dependent on its ability to make DSBs or homologous recombination proteins. Intriguingly, SUN1, a protein required for telomere attachment to the nuclear envelope and for post-DSB synapsis, is also required for early pre-DSB homolog pairing. Furthermore, pre-DSB pairing at telomeres persists upon entry into prophase I and is most likely important for initiation of synapsis. Our findings suggest that the DSB-triggered homology search may mainly serve to proofread and stabilize the pre-DSB pairing of homologous chromosomes.

Sternal osteomyelitis caused by Aspergillus fumigatus following cardiac surgery: Case and review.

Postsurgical sternal wound infection is a serious post-operative complication of cardiac surgery. Aspergillus infection of the sternum is extremely rare. We describe a case of sternal infection due to Aspergillus in an immunocompetent patient following aortic valve replacement.

The radially swollen 4 separase mutation of Arabidopsis thaliana blocks chromosome disjunction and disrupts the radial microtubule system in meiocytes.

The caspase-family protease, separase, is required at the onset of anaphase to cleave the cohesin complex that joins replicated sister chromatids. However, in various eukaryotes, separase has acquired additional and distinct functions. A single amino-acid substitution in separase is responsible for phenotypes of the Arabidopsis thaliana mutant, radially swollen 4 (rsw4). This is a conditional mutant, resembling the wild type at the permissive temperature (∼20°C) and expressing mutant phenotypes at the restrictive temperature (∼30°C). Root cells in rsw4 at the restrictive temperature undergo non-disjunction and other indications of the loss of separase function. To determine to what extent separase activity remains at 30°C, we examined the effect of the mutation on meiosis, where the effects of loss of separase activity through RNA interference are known; and in addition, we examined female gametophyte development. Here, we report that, at the restrictive temperature, replicated chromosomes in rsw4 meiocytes typically fail to disjoin and the cohesin complex remains at centromeres after metaphase. Meiotic spindles appear normal in rsw4 male meiocytes; however the mutation disrupts the radial microtubule system, which is replaced by asymmetric arrays. Surprisingly, female gametophyte development was relatively insensitive to loss of separase activity, through either rsw4 or RNAi. These effects confirm that phenotypes in rsw4 result from loss of separase activity and establish a role for separase in regulating cell polarization following male meiosis.

Classification of wounds at risk and their antimicrobial treatment with polihexanide: a practice-oriented expert recommendation.

Currently, there are no generally accepted definitions for wounds at risk of infection. In clinical practice, too many chronic wounds are regarded as being at risk of infection, and therefore many topical antimicrobials - in terms of frequency and duration of use - are applied to wounds. Based on expert discussion and current knowledge, a clinical assessment score was developed. The objective of this wounds at risk (W.A.R.) score is to allow decision-making on the indication for the use of antiseptics on the basis of polihexanide. The proposed clinical classification of W.A.R. shall facilitate the decision for wound antisepsis and allow an appropriate general treatment regimen with the focus on the prevention of wound infection. The W.A.R. score is based on a clinically oriented risk assessment using concrete patient circumstances. The indication for the use of antiseptics results from the addition of differently weighted risk causes, for which points are assigned. Antimicrobial treatment is justified in the case of 3 or more points.

The expression profile of the major mouse SPO11 isoforms indicates that SPO11beta introduces double strand breaks and suggests that SPO11alpha has an additional role in prophase in both spermatocytes and oocytes.

Both in mice and humans, two major SPO11 isoforms are generated by alternative splicing: SPO11alpha (exon 2 skipped) and SPO11beta. Thus, the alternative splicing event must have emerged before the mouse and human lineages diverged and was maintained during 90 million years of evolution, arguing for an essential role for both isoforms. Here we demonstrate that developmental regulation of alternative splicing at the Spo11 locus governs the sequential expression of SPO11 isoforms in male meiotic prophase. Protein quantification in juvenile mice and in prophase mutants indicates that early spermatocytes synthesize primarily SPO11beta. Estimation of the number of SPO11 dimers (betabeta/alphabeta/alphaalpha) in mutants in which spermatocytes undergo a normal number of double strand breaks but arrest in midprophase due to inefficient repair argues for a role for SPO11beta-containing dimers in introducing the breaks in leptonema. Expression kinetics in males suggested a role for SPO11alpha in pachytene/diplotene spermatocytes. Nevertheless, we found that both alternative transcripts can be detected in oocytes throughout prophase I, arguing against a male-specific function for this isoform. Altogether, our data support a role for SPO11alpha in mid- to late prophase, presumably acting as a topoisomerase, that would be conserved in male and female meiocytes.

Arabidopsis separase functions beyond the removal of sister chromatid cohesion during meiosis.

Separase is a capase family protease that is required for the release of sister chromatid cohesion during meiosis and mitosis. Proteolytic cleavage of the alpha-kleisin subunit of the cohesin complex at the metaphase-to-anaphase transition is essential for the proper segregation of chromosomes. In addition to its highly conserved role in cleaving the alpha-kleisin subunit, separase appears to have acquired additional diverse activities in some organisms, including involvement in mitotic and meiotic anaphase spindle assembly and elongation, interphase spindle pole body positioning, and epithelial cell reorganization. Results from the characterization of Arabidopsis (Arabidopsis thaliana) separase (ESP) demonstrated that meiotic expression of ESP RNA interference blocked the proper removal of cohesin from chromosomes and resulted in the presence of a mixture of fragmented chromosomes and intact bivalents. The presence of large numbers of intact bivalents raised the possibility that separase may also have multiple roles in Arabidopsis. In this report, we show that meiotic expression of ESP RNA interference blocks the removal of cohesin during both meiosis I and II, results in alterations in nonhomologous centromere association, disrupts the radial microtubule system after telophase II, and affects the proper establishment of nuclear cytoplasmic domains, resulting in the formation of multinucleate microspores.

SWI1 is required for meiotic chromosome remodeling events.

The Arabidopsis dsy10 mutant was previously identified as being defective in the synapsis of meiotic chromosomes resulting in male and female sterility. We report here the molecular analysis of the mutation and show that it represents a T-DNA insertion in the third exon of the SWI1 gene. Four mutations have now been identified in SWI1, several of which exhibit different phenotypes. For example, the swi1-1 and dyad mutations only affect meiosis in megasporocytes, while the swi1-2 and dsy10 mutations block both male and female meiosis. Furthermore, as part of a detailed cytological characterization of dsy10 meiocytes, we identified several differences during male meiosis between the swi1-2 and dys10 mutants, including variations in the formation of axial elements, the distribution of cohesin proteins and the timing of the premature loss of sister chromatid cohesion. We demonstrate that dsy10 represents a complete loss-of-function mutation, while a truncated form of SWI1 is expressed during meiosis in swi1-2 plants. We further show that dys10 meiocytes exhibit alterations in modified histone patterns, including acetylated histone H3 and dimethylated histone H3-Lysine 4.