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BACKGROUND: Different strategies to boost cellular NAD+ levels, such as supplementation of NAD+ precursors, or inhibition of NAD+ consumption, are currently under investigation as promising means to promote healthy aging and ameliorate dysfunctional metabolism. CD38 is a NAD+-dependent enzyme that converts NAD+ to different Ca2+-active second messengers, involved in the regulation of different signaling pathways, cell functions and metabolism. TRPM2, is an ion channel that allows Ca2+ influx from the extracellular space toward the cytosol, and is gated by ADPR, one of the molecules produced from the NAD+ degradation. In the context of systemic energy metabolism, brown adipocytes, the parenchymal cells of brown adipose tissue (BAT) as well as beige adipocytes that emerge in white adipose tissue (WAT) depots in response to catabolic conditions, are important to maintain metabolic homeostasis, together with liver. HYPOTHESIS: We aim to understand the functional relevance of CD38 and TRPM2 in the regulation of energy metabolism and NAD(P)(H) levels in BAT, WAT and liver during thermogenesis. METHODS: We used wild type, Cd38-/- and Trpm2-/- mice, exposed to cold temperatures and BAT, WAT and liver were collected. We evaluated mRNA levels by RT-PCR, proteins/enzymes levels by Western blot, FACS analysis and enzymatic activities. NAD(P)(H) levels were determined with cycling assays. Furthermore, we performed a set of in vivo experiments in which O2 consumption, CO2 production and energy expenditure were measured in mice upon thermogenic stimulation. RESULTS: We confirmed that CD38 is a major NAD+-consumer in BAT, WAT and liver: increased NAD+ levels were observed in these tissues from Cd38-/- compared with wild type mice. Interestingly, during cold exposure, a marked downregulation of CD38 expression (as detected at the mRNA, protein and enzymatic level) occurred in BAT, WAT and in liver of wild type mice. As a consequence of CD38 downregulation, an increase in NAD+ levels occurred in BAT. Instead, in WAT, CD38 downregulation was accompanied by a strong increase in NADP(H) levels, likely as a consequence of increased NADK, G6PD and malic enzyme activities. In liver, CD38 downregulation was paralleled by increased NAD(H) levels. NADK activity and NADP+ levels were not significantly modified in liver during cold-exposure. Notably, a marked decrease of NADPH level occurred in liver from both wild type and Cd38-/- mice exposed to a cold temperature, possibly as a consequence of the observed downregulation of the hepatic G6PD activity. Saving G6P from the PPP is in line with the increased activity of the enzyme glucose-6-phosphatase in liver of wild type, but not Cd38-/-, cold-exposed mice, with up-regulated gluconeogenesis. When Cd38-/- mice were kept at 6�C, higher levels of Ucp1 and Pgc-1? in BAT and WAT were revealed, compared with wild type mice. Conversely, when Trpm2-/- mice were exposed to cold temperature, lower levels of these two browning marker genes were detected, compared with wild type mice. In line with this, mice lacking Trpm2 displayed lower respiration rate and energy expenditure, when thermogenesis was induced by cold exposure and CL316,243 (a specific compound triggering adipose tissue activation). Interestingly, during cold exposure, a marked Trpm2 overexpression was observed in WAT and BAT of wild type mice. In addition, ADPR levels and mono/poly-ADPR hydrolases expression were higher in mice exposed to cold, in comparison with wild type mice. CONCLUSION: Taken together, these results demonstrate that CD38, by modulating cellular NAD(P)+ levels, is involved in the regulation of thermogenic responses in cold-activated BAT and WAT. Indeed, CD38 inhibition is being investigated as a possible strategy to ameliorate dysfunctional metabolism, by boosting NAD+ levels and sirtuins? activity. In addition, TRPM2 plays a pivotal role in BAT and WAT activation. TRPM2 gating, is likely due to alternative pathways that do not include CD38 activity.

NAPRT, the rate-limiting enzyme of the Preiss–Handler NAD biosynthetic pathway, has emerged as a key biomarker for the clinical success of NAMPT inhibitors in cancer treatment. Previous studies found that high protein levels of NAPRT conferred resistance to NAMPT inhibition in several tumor types whereas the simultaneous blockade of NAMPT and NAPRT results in marked anti-tumor effects. While research has mainly focused on NAMPT inhibitors, the few available NAPRT inhibitors (NAPRTi) have a low affinity for the enzyme and have been scarcely characterized. In this work, a collection of diverse compounds was screened in silico against the NAPRT structure, and the selected hits were tested through cell-based assays in the NAPRT-proficient OVCAR-5 ovarian cell line and on the recombinant hNAPRT. We found different chemotypes that efficiently inhibit the enzyme in the micromolar range concentration and for which direct engagement with the target was verified by differential scanning fluorimetry. Of note, the therapeutic potential of these compounds was evidenced by a synergistic interaction between the NAMPT inhibitor FK866 and the new NAPRTi in terms of decreasing OVCAR-5 intracellular NAD levels and cell viability. For example, compound IM29 can potentiate the effect of FK866 of more than two-fold in reducing intracellular NAD levels. These results pave the way for the development of a new generation of human NAPRTi with anticancer activity. European Union. H2020 813284 Associazione Italiana per la Ricerca sul Cancro IG#22098 Italian Ministry of Health PE-2016-02362694, PE-2016-02363073 Ministerio de Ciencia e Innovación PID2020-116460RB-I00

Additional file 1:. Figure S1. Gating strategy for flow cytometry analysis of DCs

Background: Meniscal allograft transplantation (MAT) has produced good to excellent results in the general population; however, few investigations have examined MAT in athletes and sport-related outcomes. Purpose: To report midterm clinical outcomes of MAT and the rate of return to sport in a physically active population. Study Design: Case series; Level of evidence, 4. Methods: The study included all physically active patients who underwent arthroscopic MAT without bone plugs and had a minimum of 2 years of follow-up at a single institution. Clinical evaluation was performed with the Knee injury and Osteoarthritis Outcome Score (KOOS), the Tegner activity scale, and a 0- to 100-point subjective scale for knee function and satisfaction. Outcomes evaluated included ability to return to sport, time to return to sport, level of sport activity upon return compared with preinjury level, and level of decrease in sport participation or reasons for not returning to sport participation. Comparisons were made between patients who did or did not return to sport and between patients who returned to the same level or a decreased level. Regression analysis was performed to determine the variables affecting the outcomes. Results: Eighty-nine patients, whose mean ± SD age at surgery was 38.5 ± 11.2 years, were evaluated to a mean follow-up of 4.2 ± 1.9 years. Total KOOS improved from a mean ± SD of 39.5 ± 18.5 preoperatively to 84.7 ± 14.8 at the latest follow-up (P

The patellofemoral joint, due to its particular bone anatomy and the numerous capsuloligamentous structures and muscles that act dynamically on the patella, is considered one of the most complex joints in the human body from the biomechanical point of view. The medial patellofemoral ligament (MPFL) has been demonstrated to contribute 60% of the force that opposes lateral displacement of the patella, and MPFL injury results in an approximately 50% reduction in the force needed to dislocate the patella laterally with the knee extended. For this reason, recent years have seen a growing interest in the study of this important anatomical structure, whose aponeurotic nature has thus been demonstrated. The MPFL acts as a restraint during motion, playing an active role under conditions of laterally applied stress, but an only marginal role during natural knee flexion. However, it remains extremely difficult to clearly define the anatomy of the MPFL and its relationships with other anatomical structures.

Additional file 7: Supplementary Table 1. QPCR primer list.

Additional file 7: Supplementary Table 1. QPCR primer list.

Purpose: To present the results of a survival analysis of a series of 147 arthroscopic MAT procedures. Methods: One-hundred and forty-seven patients (117 males and 30 females) underwent arthroscopic MAT without bone plugs (82 medial MAT and 65 lateral MAT) using fresh-frozen, non-irradiated grafts. They were retrospectively reviewed at a mean of 4.0 ± 1.9-year follow-up. Mean age at surgery was 40.9 ± 11.2 (range 16.7–68.8) years; 70 patients (48 %) underwent combined procedures. Clinical evaluation was performed with KOOS, Lysholm and a 0–100 VAS for pain. Survival analysis was performed using two endpoints: surgical failure (revision procedure with direct relation to MAT) and clinical failure (revision procedure or poor Lysholm score, 50 or 25. Conclusions: MAT, eventually associated with other needed procedures, was able to significantly relieve pain and improve function of the knee joint at midterm follow-up, with a survival rate from 9.7 to 8.0 years based on failure criteria. Most additional procedures were done in the first 2 post-operative years. MAT eventually associated with other needed procedures could represent an effective treatment for post-meniscectomy syndrome. Level of evidence: Therapeutic study, retrospective case series, Level IV.

The medial patellofemoral ligament (MPFL) has been demonstrated to be the major soft tissue restraint to lateral patellar displacement; therefore its reconstruction is becoming the most performed procedure in cases of patellar dislocation. However, various alternatives have been proposed, aimed at reconstructing or reinforcing other ligamentous structures that are believed to play a crucial role in patellar stability. The reconstruction of the medial patellotibial ligament has been performed in our center, with generally good and promising results reported, and does not substantially differ from more popular surgical techniques such as MPFL reconstruction. The good results, combined with relevant technical issues such as its simple, less-invasive approach and low risk of complications, could make the technique a feasible alternative to the classic procedures of MPFL reconstruction for patellar stabilization.

At the current state of the art in anterior cruciate ligament (ACL) reconstruction, multiple techniques have been presented but none has given clearly defined and improved results.One of the main issues concerns the choice of graft. The concept of using xenograft tissue, defined as a graft tissue from one species and destined for implantation in an unlike species, was introduced in order to try to overcome the mechanical and biological concerns associated with synthetic materials and the safety and quality concerns and availability problems of allograft tissue. Xenograft tissue carries the risk of producing an immunological reaction. In order to try to overcome or attenuate the immune response against porcine xenograft tissue, the Z-Process® (Aperion Biologics Inc, San Antonio, Texas, USA) has been developed and used to produce the Z-Lig® family of devices for ACL reconstruction procedures. Z-Lig® is a tendon graft with or without bone blocks, sourced from animal tissue in a manner consistent with what has normally been sourced from human tissue, and processed to overcome anti-Gal-mediated rejection and to attenuate other immunological recognition in humans. All this while ensuring sterility, viral inactivation and preservation of mechanical proprieties appropriate for an ACL reconstruction device. The Z-Lig® device has been tested in skeletally mature monkeys and given interesting and promising results from the preclinical performance and safety profile point of view. On this basis, it was possible to proceed with the first clinical trial involving humans, which gave similar encouraging results.The Z-Lig® device has also been implanted in Italy at the Rizzoli Orthopaedic Institute in Bologna, as a part of international multicenter prospective randomized blinded controlled study aimed at comparing xenograft with allograft tissue.

PURPOSE: The purpose of this systematic review was to summarise and evaluate the clinical outcomes of the collagen meniscus implant (CMI) and its complication and failure rates. These data were then used to evaluate the results of the CMI at different follow-up time periods and investigate possible differences in the behaviour of lateral and medial CMI. METHODS: A comprehensive search was performed in PubMed, MEDLINE, CINAHL, Cochrane, EMBASE and Google Scholar databases using various combinations of the following keywords: "collagen meniscus implant" or "collagen meniscal implant". All studies evaluating medial or lateral CMI using the Lysholm score, visual analogue scale (VAS) for pain, Tegner activity scale and subjective or objective International Knee Documentation Committee (IKDC) scores were included in the systematic review. RESULTS: Eleven studies were included in the systematic review. The pooled number of patients involved in CMI surgery were 396 (90.2 % medial, 9.8 % lateral), with a mean age at surgery of 37.8 years. Concomitant procedures were present in 48.8 % of patients; most of them were anterior cruciate ligament (ACL) reconstruction, high tibial osteotomy (HTO) and microfractures. The Lysholm score and VAS for pain showed an improvement at six months up to ten years. No noticeable differences were present comparing short-term values of Lysholm score between medial and lateral CMI. The Tegner activity level reached its peak at 12 months after surgery and showed a progressive decrease through five and ten years post CMI implantation, however always remaining above the pre-operative level. Only a few knees were rated as "nearly abnormal" or "abnormal" at IKDC grading at all follow-up evaluations. CONCLUSIONS: The CMI could produce good and stable clinical results, particularly regarding knee function and pain, with low rates of complications and reoperations.

PURPOSE: A deficiency of posterolateral structures significantly increases the varus load on the ACL, while a chronic ACL lesion, the increased tibial rotation and the repetitive non-physiological knee motion, could affect and damage the integrity of the popliteus tendon. Therefore, the aim of the present study was to report the very long clinical outcomes of a combined single-bundle BPTB ACL reconstruction and popliteus plasty according to Bousquets technique, for the treatment of combined chronic anterior and posterolateral laxities. METHODS: Fifteen patients that underwent combined ACL reconstruction and popliteal plasty according to Bousquets technique were available at mean 26.8 ± 1.0 years (range 25.4-28.0 years). All the patients were evaluated clinically and 13 by means of KT-1000 Arthrometer as well. Subjective evaluation was performed with the subjective IKDC, WOMAC and a 0-10 VAS for pain scales. RESULTS: At clinical evaluation, 10 patients (67 %) presented a negative anterior drawer test; Lachman test was negative in nine patients (60 %); the varus stress test was negative in eight (53 %); and the dial test was negative in all but one patient (93 %). Only two patients (15 %) presented a side-to-side difference >5 mm at the instrumented laxity evaluation. CONCLUSION: The combined single-bundle BPTB ACL reconstruction and popliteal plasty according to Bousquets technique were able to produce very good long-term results, in terms of knee stability, subjective outcomes, functional results and return to sport activity, in case of chronic anterior and posterolateral laxities. LEVEL OF EVIDENCE: Retrospective case series, Level IV.

To evaluate mid-term clinical and radiographic outcomes after an original medial patellotibial ligament reconstruction in patients with patellar dislocation. METHODS: Twenty-nine knees (27 patients, 8 males and 19 females) treated for patellar dislocation with medialization of the patellar tendon medial third combined with medial and lateral release were evaluated clinically and radiographically at a mean follow-up of 6.1±2.5 years. Trochleoplasty was performed in case of severe flat trochlea (6 knees, 21%). Aetiology of patellofemoral instability was traumatic in 6 (21%) and atraumatic in 23 (79%) knees. The mean age at first dislocation was 19.2±10.1 years. WOMAC, subjective and objective IKDC, Kujala, VAS for pain, Tegner activity and EQ-5D scores were used. Anteroposterior, lateral and 30° axial views were performed for radiographic monitoring. RESULTS: There was a significant improvement of all clinical scores and significant reduction in knee pain. Twenty-four knees (83%) were normal or nearly normal by objective IKDC score at final follow-up. Radiographs showed a higher incidence of patella alta and flat trochlea in the atraumatic group. Severe signs of patellar osteoarthritis were found in 1 knee (3%). A higher body mass index (BMI) was correlated with worse pre-operative scores. Four knees (14%) were considered failures (2 further dislocations, 2 revision surgeries). The overall survival rate at 6 years was 0.811. CONCLUSIONS: The presented techniques produced good clinical and radiographic results at mean 6.1 years follow-up, with 14 % failures. Signs of patellofemoral dysplasia were found in patients with atraumatic patellar dislocation. BMI was related to worse pre-operative clinical status. LEVEL OF EVIDENCE: Retrospective study, Level IV.