Casein kinase 2 phosphorylates and induces the SALL2 tumor suppressor degradation in colon cancer cells.

Spalt-like proteins are Zinc finger transcription factors from Caenorhabditis elegans to vertebrates, with critical roles in development. In vertebrates, four paralogues have been identified (SALL1-4), and SALL2 is the family's most dissimilar member. SALL2 is required during brain and eye development. It is downregulated in cancer and acts as a tumor suppressor, promoting cell cycle arrest and cell death. Despite its critical functions, information about SALL2 regulation is scarce. Public data indicate that SALL2 is ubiquitinated and phosphorylated in several residues along the protein, but the mechanisms, biological consequences, and enzymes responsible for these modifications remain unknown. Bioinformatic analyses identified several putative phosphorylation sites for Casein Kinase II (CK2) located within a highly conserved C-terminal PEST degradation motif of SALL2. CK2 is a serine/threonine kinase that promotes cell proliferation and survival and is often hyperactivated in cancer. We demonstrated that CK2 phosphorylates SALL2 residues S763, T778, S802, and S806 and promotes SALL2 degradation by the proteasome. Accordingly, pharmacological inhibition of CK2 with Silmitasertib (CX-4945) restored endogenous SALL2 protein levels in SALL2-deficient breast MDA-MB-231, lung H1299, and colon SW480 cancer cells. Silmitasertib induced a methuosis-like phenotype and cell death in SW480 cells. However, the phenotype was significantly attenuated in CRISPr/Cas9-mediated SALL2 knockout SW480 cells. Similarly, Sall2-deficient tumor organoids were more resistant to Silmitasertib-induced cell death, confirming that SALL2 sensitizes cancer cells to CK2 inhibition. We identified a novel CK2-dependent mechanism for SALL2 regulation and provided new insights into the interplay between these two proteins and their role in cell survival and proliferation.

Role of HDAC6-STAT3 in immunomodulatory pathways in Colorectal cancer cells.

Colorectal cancer (CRC) is one of the most common malignant neoplasms and the second leading cause of death from tumors worldwide. Therefore, there is a great need to study new therapeutical strategies, such as effective immunotherapies against these malignancies. Unfortunately, many CRC patients do not respond to current standard immunotherapies, making it necessary to search for adjuvant treatments. Histone deacetylase 6 (HDAC6) is involved in several processes, including immune response and tumor progression. Specifically, it has been observed that HDAC6 is required to activate the Signal Transducer and Activator of Transcription 3 (STAT3), a transcription factor involved in immunogenicity, by activating different genes in these pathways, such as PD-L1. Over-expression of immunosuppressive pathways in cancer cells deregulates T-cell activation. Therefore, we focused on the pharmacological inhibition of HDAC6 in CRC cells because of its potential as an adjuvant to avoid immunotolerance in immunotherapy. We investigated whether HDAC6 inhibitors (HDAC6is), such as Nexturastat A (NextA), affected STAT3 activation in CRC cells. First, we found that NextA is less cytotoxic than the non-selective HDACis panobinostat. Then, NextA modified STAT3 and decreased the mRNA and protein expression levels of PD-L1. Importantly, transcriptomic analysis showed that NextA treatment affected the expression of critical genes involved in immunomodulatory pathways in CRC malignancies. These results suggest that treatments with NextA reduce the functionality of STAT3 in CRC cells, impacting the expression of immunomodulatory genes involved in the inflammatory and immune responses. Therefore, targeting HDAC6 may represent an interesting adjuvant strategy in combination with immunotherapy.

Treatment of Large Cartilage Defects in the Knee by Hydrogel-Based Autologous Chondrocyte Implantation: Two-Year Results of a Prospective, Multicenter, Single-Arm Phase III Trial.

OBJECTIVE: To evaluate the clinical outcome of a hydrogel-based autologous chondrocyte implantation (ACI) for large articular cartilage defects in the knee joint. DESIGN: Prospective, multicenter, single-arm, phase III clinical trial. ACI was performed in 100 patients with focal full-thickness cartilage defects ranging from 4 to 12 cm2 in size. The primary outcome measure was the responder rate at 2 years using the Knee Injury and Osteoarthritis Outcome Score (KOOS). RESULTS: Two years after ACI treatment, 93% of patients were KOOS responders having improved by ≥10 points compared with their pre-operative level. The primary endpoint of the study was met and demonstrated that the KOOS response rate is markedly greater than 40% with a lower 95% CI (confidence interval) of 86.1, more than twice the pre-specified no-effect level. KOOS improvement (least squares mean) was 42.0 ± 1.8 points (95% CI between 38.4 and 45.7). Mean changes from baseline were significant in the overall KOOS and in all 5 KOOS subscores from Month 3 (first measurement) to Month 24 (inclusive) (P < 0.0001). The mean MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) score after 24 months reached 80.0 points (95% CI: 70.0-90.0 points) and 92.1 points in lesions ≤ 5 cm2. CONCLUSIONS: Overall, hydrogel-based ACI proved to be a valuable treatment option for patients with large cartilage defects in the knee as demonstrated by early, statistically significant, and clinically meaningful improvement up to 2 years follow-up. Parallel to the clinical improvements, MRI analyses suggested increasing maturation, re-organization, and integration of the repair tissue. TRIAL REGISTRATION: NCT03319797; EudraCT No.: 2016-002817-22.

Streamlined computational pipeline for genetic background characterization of genetically engineered mice based on next generation sequencing data.

BACKGROUND: Genetically engineered mice (GEM) are essential tools for understanding gene function and disease modeling. Historically, gene targeting was first done in embryonic stem cells (ESCs) derived from the 129 family of inbred strains, leading to a mixed background or congenic mice when crossed with C57BL/6 mice. Depending on the number of backcrosses and breeding strategies, genomic segments from 129-derived ESCs can be introgressed into the C57BL/6 genome, establishing a unique genetic makeup that needs characterization in order to obtain valid conclusions from experiments using GEM lines. Currently, SNP genotyping is used to detect the extent of 129-derived ESC genome introgression into C57BL/6 recipients; however, it fails to detect novel/rare variants. RESULTS: Here, we present a computational pipeline implemented in the Galaxy platform and in BASH/R script to determine genetic introgression of GEM using next generation sequencing data (NGS), such as whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. The pipeline includes strategies to uncover variants linked to a targeted locus, genome-wide variant visualization, and the identification of potential modifier genes. Although these methods apply to congenic mice, they can also be used to describe variants fixed by genetic drift. As a proof of principle, we analyzed publicly available RNA-Seq data from five congenic knockout (KO) lines and our own RNA-Seq data from the Sall2 KO line. Additionally, we performed target validation using several genetics approaches. CONCLUSIONS: We revealed the impact of the 129-derived ESC genome introgression on gene expression, predicted potential modifier genes, and identified potential phenotypic interference in KO lines. Our results demonstrate that our new approach is an effective method to determine genetic introgression of GEM.

Influence of ionizing radiation on the antimicrobial activity of the antibiotics sulfamethoxazole and trimethoprim.

The response of the antimicrobial compounds sulfamethoxazole (SMX) and trimethoprim (TMP) - individually and in mixtures - to ionizing radiation was investigated using laboratory prepared mixtures and a commercial pharmaceutical formulation. The residual antibacterial activity of the solutions was monitored using Staphylococcus aureus and Escherichia coli test strains. Based on antibacterial activity, SMX was more susceptible to ionizing radiation as compared to TMP. The antibacterial activity of SMX and TMP was completely eliminated at 0.2 kGy and 0.8 kGy, respectively. However, when SMX and TMP were in a mixture, the dose required to eliminate the antibacterial activity was 10 kGy, implying a synergistic antibacterial activity when these are present in mixtures. Only when the antibiotic concentration was below the Minimum Inhibitory Concentration of TMP (i.e., 2 µmol dm-3) did the antibacterial activity of the SMX and TMP mixture disappear. These results imply that the synergistic antimicrobial activity of antimicrobial compounds in pharmaceutical waste streams is a strong possibility. Therefore, antimicrobial activity assays should be included when evaluating the use of ionizing radiation technology for the remediation of pharmaceutical or municipal waste streams.

Sall2 is required for proapoptotic Noxa expression and genotoxic stress-induced apoptosis by doxorubicin.

The Sall2 transcription factor is deregulated in several cancers; however, little is known about its cellular functions, including its target genes. Recently, we demonstrated that p53 directly regulates Sall2 expression under genotoxic stress. Here, we investigated the role of Sall2 in the context of cellular response to genotoxic stress. In addition, we further examined the Sall2-p53 relationship during genotoxic stress in primary mouse embryo fibroblasts (MEFs), which are derived from Sall2 knockout mice separately, or in combination with the p53ERTAM knock-in mice. We found that the levels of Sall2 mRNA and protein are dynamically modulated in response to doxorubicin. At early times of stress, Sall2 is downregulated, but increases under extension of the stress in a p53-independent manner. Based on caspase-3/7 activities, expression of cleaved poly (ADP-ribose) polymerase, expression of cleaved caspase-3 and induction of proapoptotic proteins, Sall2 expression was correlated with cellular apoptosis. Consequently, Sall2-/- MEFs have decreased apoptosis, which relates with increased cell viability in response to doxorubicin. Importantly, Sall2 was required for apoptosis even in the presence of fully activated p53. Searching for putative Sall2 targets that could mediate its role in apoptosis, we identified proapoptotic NOXA/PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1). We demonstrated that Sall2 positively regulates Noxa promoter activity. Conserved putative Sall2-binding sites at the NOXA promoter were validated in vitro by electrophoretic mobility shift assay and in vivo by ChIP experiments, identifying NOXA as a novel Sall2 target. In agreement, induction of Noxa protein and mRNA in response to doxorubicin was significantly decreased in Sall2-/- MEFs. In addition, studies in leukemia Jurkat T cells support the existence of the Sall2/Noxa axis, and the significance of this axis on the apoptotic response to doxorubicin in cancer cells. Our study highlights the relevance of Sall2 in the apoptotic response to extended genotoxic stress, which is important for understanding its role in normal physiology and disease.

Promoting secure attachment: evaluation of the effectiveness of an early intervention pilot programme with mother-infant dyads in Santiago, Chile.

BACKGROUND: Research indicates that the early attachment patterns of babies could influence their socio-emotional development and prevent the emergence of problematic behaviours in the child later in life. Many studies in the field of early attachment interventions have promoted a secure attachment bond between mother and infant. The purpose of this study was to evaluate the effectiveness of an early pilot intervention programme designed to promote a secure attachment bond in mother-infant dyads belonging to a population seeking regular treatment at urban health centres in Santiago, Chile. METHODS: Primipara mothers were randomly assigned to two intervention conditions: a secure attachment promotion programme (experimental group = 43) or an educational talk (control group = 29). The Strange Situation Assessment was used to collect data on the attachment patterns of babies. RESULTS: The results show that after the intervention, there were more babies with secure attachment in the experimental group than in the control group. CONCLUSIONS: These findings represent a preliminary step towards evaluating interventions aimed at promoting secure attachment in Chilean mother-child dyads. While the effect of the intervention is not significant, the effect size obtained is respectable and consistent with other meta-analytic findings.

Body composition after endogenous (Cushing's syndrome) and exogenous (rheumatoid arthritis) exposure to glucocorticoids.

Exposure to chronic glucocorticoid (GC) excess determines changes in body composition. The aim of the study was to compare body composition in women exposed to endogenous hypercortisolism (Cushing's syndrome, CS), exogenous glucocorticoid treatment (rheumatoid arthritis, RA) and controls. Fifty-one CS women, 26 RA women treated with low-dose prednisone (5 mg/day or 10 mg/2 days), and 78 female controls were included. Fourteen CS patients were hypercortisolemic, 37 in remission (10 required hydrocortisone substitution after surgery). Body composition parameters were measured by dual-energy X-ray absorptiometry scanning (DEXA). RA patients had a greater waist-hip ratio (WHR) (p<0.01), less lean body mass (LBM) (p<0.01), and lumbar bone mineral density (BMD) (p<0.01) than controls. CS patients, globally and those with cured disease, had more total fat (both percentage and kg) and trunk fat percentage, and less whole body-BMD than RA patients (p<0.05, p<0.01, p<0.05, respectively). Active CS patients had less whole body-BMD and more LBM than RA patients (p<0.05, p=0.01, respectively). Cured CS patients not taking hydrocortisone had more total fat [both percentage (p<0.05) and kg (p<0.05)], trunk fat percentage (p<0.05), lumbar BMD (p<0.01) than RA patients. Cured CS patients requiring hydrocortisone only differed from RA patients by smaller WHR (p<0.01). All the differences in BMD disappeared when the data were reanalyzed including only the estrogen-deficient groups. Hypercortisoliof CS determines an irreversible increase in body fat, greater than in RA. Endogenous and exogenous exposure to GC negatively affects body composition by increasing the WHR. There appears to be no additional effect on BMD in estrogen-deficient women.

The importance of arthroscopy in diagnosing synovial haemangioma of the knee joint.

In connection with one of their cases, authors call attention to the importance of differential diagnosis in synovial haemangioma of the knee joint, a rare condition. They also give a survey of its clinical, radiological and pathological features, review the literature on the topic, with special emphasis on the importance of arthroscopy in making the clinical diagnosis, and describe the treatment applied in their case.

Functional improvement after knee arthroplasty without resurfacing of patella.

There has been no universal agreement so far regarding the necessity of patellar resurfacing in total knee arthroplasty. As resurfacing has been reported to be associated with high incidence of complications, this practice has been avoided in our Department. A report is given on the analysis of the functional outcome of 60 knee arthroplasties without patellar resurfacing in 53 patients (7 bilateral) followed up for twelve to thirty months, with special regard to the functions closely related to patelloformal articulation. The underlying diagnosis was osteoarthritis in 78.3%, rheumatoid arthritis in 13.3%, and posttraumatic arthritis in 8.3% of the patients. Graded according to the modified knee-rating system of the Hospital for Special Surgery, excellent or good results were obtained in the case of 55 knees (91.6%) and the mean score improved from 53.6 points preoperatively to 82.6 points following arthroplasty. Subjective and objective functional assessment of stair climbing and transfer activities have shown no functional deficit attributed to the patellofemoral joint of the replaced knee.

Identification of sites of injury in Lactobacillus bulgaricus during heat stress.

Heat resistance of Lactobacillus bulgaricus in skimmed milk at 62 degrees, 64 degrees, 65 degrees and 66 degrees C was studied. The response to increasing temperatures in this range was not linear, with temperatures at 65 degrees C and above giving a lower survival rate than would be predicted from experiments at lower temperatures. To identify sites of injury at these temperatures, chemical markers were used. Heating at 64 degrees C and below resulted in damage to the cytoplasmic membrane. At temperatures of 65 degrees C and above chemical markers also indicated damage in the cell wall and proteins. Using differential scanning calorimetry analysis of whole cells of Lact. bulgaricus seven main peaks were observed (1-51, m1-61, m2-73, n-80, p-89, q-100, r-112 degrees C). Three of these peaks (l(r), m(r) and p(r)) were the result of reversible reactions. Analysis of cell fractions identified the cell structure involved in giving rise to each of the three reversible peaks; l(r), cell membrane lipids, m(r), ribosomes, and p(r), DNA. The evidence presented in this paper shows that irreversible reactions in the cell ribosomes are a critical site of damage in Lact. bulgaricus during heat stress in liquid media at 65 degrees C and above.

The pathological plica in the knee.

Fourteen patients were evaluated who had an arthroscopic removal of the symptomatic mediopatellar plica of the knee in this retrospective study. The main complaint of the patients was the pain at the medial side of the knee. Before the operation only in 3 cases was the diagnosis the pathological plica. The arthroscopy was performed in a routine way. The plica was consider pathological, and removed, when it was thickened. There was grade I or II chondropathy in the knee on the medial femoral condyle and the patella respectively, caused by the plica. Good to excellent result were obtained in 93% of the knee with or without chondromalacia.

Acute arthroscopy.

The role and significance of acute arthroscopy have been evaluated in the treatment of knee joint injuries on the basis of findings during 59 arthroscopic operations which were conducted within two weeks after the accident. The injuries developed isolated in more than half of the cases (65%) whereas they appeared in a combination of two or more in 26% and in 9%, respectively. Injuries requiring operation were found in 91.5%, most of which were ruptures of the ACL (33 cases) and menisci (23 cases). In the case of ACL rupture, in the acute phase on sportsmen and physical workers primary arthroscopically assisted transligamental replacement was performed with patellar graft while in the case of proximal rupture of the ACL reinsertion and augmentation were carried out with semitendinosus tendon. The ruptures of dislocated eminentia were refixed in each case. In the case of the rupture of meniscus the refixation of the meniscus of resection of the ruptured part was attempted. By means of acute arthroscopy the lesion of the intraarticular structures or that of their combinations can be exactly diagnosed. Depending on the findings of arthroscopy the injuries can be treated immediately or operated on at a later time, thus preventing the joint from further deterioration.

Therapeutic value of continuous passive motion after anterior cruciate replacement.

The aim of this study is the evaluation of the therapeutic value of continuous passive motion after ACL replacement. After 41 ACL reconstruction in 13 cases only active motion, in 28 cases active motion and CPM were used postoperatively. Stability, the range of motion and complications were evaluated at the time of discharge, 3 and 6 months after the operation. The flexion-extension in the CPM group was significantly greater at the time of discharge, but this difference was practically eliminated in 6 months after the operation. There were not any other significant difference between the two groups. The CPM gives only a little advantage in the rehabilitation after the ACL replacement.

Managing sacral pressure ulcers with hydrocolloid dressings: results of a controlled, clinical study.

One-hundred and three patients with Stage II and III sacral pressure ulcers were enrolled in a prospective, controlled, multi-center clinical study to evaluate and compare dressing performance, safety and efficacy. Fifty-two patients were randomized to treatment with a triangle-shaped hydrocolloid border dressing and 51 patients were randomized to a different, oval shape, hydrocolloid dressing. The majority of patients (70 percent) utilized a pressure reducing mattress or bed. Most ulcers were Stage II, had existed for < 1 month and exhibited no change utilizing previous treatments. Patients and wounds were similarly distributed among treatment groups. Patients in the oval dressing group were more likely to exhibit a product related adverse reaction resulting in discontinuation of treatment as compared to patients treated with the triangle border dressing (p = 0.057, Fisher's Exact Test). Wear time was longest for wounds dressed with the triangle dressing applied point down. Incontinence reduced the interval between dressing changes in both groups. Healing was more likely to occur in wounds dressed with the triangle border dressing. These ulcers showed a greater reduction in ulcer width as compared to wounds dressed with the oval dressing (p < 0.03, Fisher's Exact Test).

Mathematical modelling of the combined effect of water activity, pH and redox potential on the heat destruction.

Heat destruction of seven foodborne microorganisms (Lactobacillus plantarum, Lactobacillus brevis, Saccharomyces cerevisiae, Zygosaccharomyces bailii, Yarrowia lipolytica, Paecilomyces varioti and Neosartoria fischeri) as a function of the temperature, pH, redox potential and water activity was studied in synthetic heating media. Several mathematical models were developed for describing the heat destruction rate, most of them resulted in a good correlation between the fitted and measured values. The determination coefficients of the model-fitting were the best in case of lactobacilli and moulds (0.96-0.99) and the worst in case of the yeasts (0.81-0.88).

[The KT 1000 arhtrometer in the comparative study of stability of knee joints operated on for instability]. / Instabilitás miatt operált térdízületek KT 1000 arthrométerrel meghatározott stabilitásának összehasonlító vizsgálata.

In 1981 through 1989 authors performed 160 complex reconstruction operations for instability of the knee joint according to the basic principles of W. Müller, striving to restore all structures injured. 92 patients with 378 operations were controlled. The assessment of the results was based on the physical examination, the measurement of ligament laxity with the K 1000 arthrometer, the opinion of the patients, the level of their activity and Lysholm's functional point system was used. The stability of the knee could be restored in 73 per cent of the cases. The patients have thought the knees, found more stable with the arthrometer, really more stable. There was however no unambiguous relation between the knee stability and the level of activity of the patients. The most frequent complaint was pain, increased synovial fluid and the hindrance of squatting. The results were better if the patient had no previous operation in an other department and in those who did not need menisectomy, who had less operations, less body weight and better muscular force.

[Measuring the looseness of the knee joint by using the Kt 1000 arthrometer]. / A térd lazaság mérése MEDmetric KT 1000 arthrotméterrel.

Authors measured the stability in the sagittal plane of the intact knee joints of 50 young men with the MEDmetric KT 1000 apparatus. The greatest movement during the measurements on 100 knees was observed in 25 degrees flexion and during the application of force 89N. In 70 degrees flexion the movement was only half of that observed in 25 degrees flexion. The difference of the average movements measured bilaterally was neither in active nor in passive experiments more than 0.5 mm. The difference between the average values of the compliance indexes on the left and right side was only 0.01 mm. In all measurements the frequency distribution of the differences between the two sides was essentially the same. The difference between the movements on the two sides did not exceed 2 mm-s in 90-100 percent of the cases.