Revolutionizing in vivo therapy with CRISPR/Cas genome editing: breakthroughs, opportunities and challenges.

Genome editing using the CRISPR/Cas system has revolutionized the field of genetic engineering, offering unprecedented opportunities for therapeutic applications in vivo. Despite the numerous ongoing clinical trials focusing on ex vivo genome editing, recent studies emphasize the therapeutic promise of in vivo gene editing using CRISPR/Cas technology. However, it is worth noting that the complete attainment of the inherent capabilities of in vivo therapy in humans is yet to be accomplished. Before the full realization of in vivo therapeutic potential, it is crucial to achieve enhanced specificity in selectively targeting defective cells while minimizing harm to healthy cells. This review examines emerging studies, focusing on CRISPR/Cas-based pre-clinical and clinical trials for innovative therapeutic approaches for a wide range of diseases. Furthermore, we emphasize targeting cancer-specific sequences target in genes associated with tumors, shedding light on the diverse strategies employed in cancer treatment. We highlight the various challenges associated with in vivo CRISPR/Cas-based cancer therapy and explore their prospective clinical translatability and the strategies employed to overcome these obstacles.

Bone mineral density (BMD) and computer tomographic measurements of the equine proximal phalanx in correlation with breaking strength.

Despite the fact that bone mineral density (BMD) is an important fracture risk predictor in human medicine, studies in equine orthopedic research are still lacking. We hypothesized that BMD correlates with bone failure and fatigue fractures of this bone. Thus, the objectives of this study were to measure the structural and mechanical properties of the proximal phalanx with dual energy X-ray absorptiometry (DXA), to correlate the data obtained from DXA and computer tomography (CT) measurements to those obtained by loading pressure examination and to establish representative region of interest (ROI) for in vitro BMD measurements of the equine proximal phalanx for predicting bone failure force. DXA was used to measure the whole bone BMD and additional three ROI sites in 14 equine proximal phalanges. Following evaluation of the bone density, whole bone, cortical width and area in the mid-diaphyseal plane were measured on CT images. Bones were broken using a manually controlled universal bone crusher to measure bone failure force and reevaluated for the site of fractures on follow-up CT images. Compressive load was applied at a constant displacement rate of 2 mm/min until failure, defined as the first clear drop in the load measurement. The lowest BMD was measured at the trabecular region (mean +/- SD: 1.52 +/- 0.12 g/cm2; median: 1.48 g/cm2; range: 1.38-1.83 g/cm2). There was a significant positive linear correlation between trabelcular BMD and the breaking strength (P = 0.023, r = 0.62). The trabecular region of the proximal phalanx appears to be the only significant indicator of failure of strength in vitro. This finding should be reassessed to further reveal the prognostic value of trabecular BMD in an in vivo fracture risk model.

Diffusion of contrast medium after four different techniques for analgesia of the proximal metacarpal region: an in vivo and in vitro study.

REASONS FOR PERFORMING STUDY: There is limited information on potential diffusion of local anaesthetic solution after various diagnostic analgesic techniques of the proximal metacarpal region. OBJECTIVE: To document potential distribution of local anaesthetic solution following 4 techniques used for diagnostic analgesia of the proximal metacarpal region. METHODS: Radiodense contrast medium was injected around the lateral palmar or medial and lateral palmar metacarpal nerves in 8 mature horses, using 4 different techniques. Radiographs were obtained 0, 10 and 20 min after injection and were analysed subjectively. A mixture of radiodense contrast medium and methylene blue was injected into 4 cadaver limbs; the location of the contrast medium and dye was determined by radiography and dissection. RESULTS: Following perineural injection of the palmar metacarpal nerves, most of the contrast medium was distributed in an elongated pattern axial to the second and fourth metacarpal bones. The carpometacarpal joint was inadvertently penetrated in 4/8 limbs after injections of the palmar metacarpal nerves from medial and lateral approaches, and in 1/8 limbs when both injections were performed from the lateral approach. Following perineural injection of the lateral palmar nerve using a lateral approach, the contrast medium was diffusely distributed in all but one limb, in which the carpal sheath was inadvertently penetrated. In 5/8 limbs, following perineural injection of the lateral palmar nerve using a medial approach, the contrast medium diffused proximally to the distal third of the antebrachium. CONCLUSIONS AND POTENTIAL RELEVANCE: Inadvertent penetration of the carpometacarpal joint is common after perineural injection of the palmar metacarpal nerves, but less so if both palmar metacarpal nerves are injected using a lateral approach. Following injection of the lateral palmar nerve using a medial approach, the entire palmar aspect of the carpus may be desensitised.

Distribution of radiodense contrast medium after perineural injection of the palmar and palmar metacarpal nerves (low 4-point nerve block): an in vivo and ex vivo study in horses.

REASONS FOR PERFORMING STUDY: Evidence-based information is limited on distribution of local anaesthetic solution following perineural analgesia of the palmar (Pa) and palmar metacarpal (PaM) nerves in the distal aspect of the metacarpal (Mc) region ('low 4-point nerve block'). OBJECTIVES: To demonstrate the potential distribution of local anaesthetic solution after a low 4-point nerve block using a radiographic contrast model. METHODS: A radiodense contrast medium was injected subcutaneously over the medial or the lateral Pa nerve at the junction of the proximal three-quarters and distal quarter of the Mc region (Pa injection) and over the ipsilateral PaM nerve immediately distal to the distal aspect of the second or fourth Mc bones (PaM injection) in both forelimbs of 10 mature horses free from lameness. Radiographs were obtained 0, 10 and 20 min after injection and analysed subjectively and objectively. Methylene blue and a radiodense contrast medium were injected in 20 cadaver limbs using the same techniques. Radiographs were obtained and the limbs dissected. RESULTS: After 31/40 (77.5%) Pa injections, the pattern of the contrast medium suggested distribution in the neurovascular bundle. There was significant proximal diffusion with time, but the main contrast medium patch never progressed proximal to the mid-Mc region. The radiological appearance of 2 limbs suggested that contrast medium was present in the digital flexor tendon sheath (DFTS). After PaM injections, the contrast medium was distributed diffusely around the injection site in the majority of the limbs. In cadaver limbs, after Pa injections, the contrast medium and the dye were distributed in the neurovascular bundle in 8/20 (40%) limbs and in the DFTS in 6/20 (30%) of limbs. After PaM injections, the contrast and dye were distributed diffusely around the injection site in 9/20 (45%) limbs and showed diffuse and tubular distribution in 11/20 (55%) limbs. CONCLUSIONS AND POTENTIAL RELEVANCE: Proximal diffusion of local anaesthetic solution after a low 4-point nerve block is unlikely to be responsible for decreasing lameness caused by pain in the proximal Mc region. The DFTS may be penetrated inadvertently when performing a low 4-point nerve block.

Diffusion of contrast medium after perineural injection of the palmar nerves: an in vivo and in vitro study.

REASONS FOR PERFORMING STUDY: Proximal diffusion of local anaesthetic solution after perineural anaesthesia may lead to the desensitisation of structures other than those intended. However, there is no evidence-based study demonstrating the potential distribution and diffusion of local anaesthetic solution after perineural analgesia in the distal limb. OBJECTIVE: To document the potential diffusion of local anaesthetic solution using a radiopaque contrast model and to evaluate the influence of walking compared with confinement in a stable after injection. METHODS: Radiopaque contrast medium was injected subcutaneously over one palmar nerve at the base of the proximal sesamoid bones in 6 nonlame mature horses. Horses were assigned randomly to stand still or walk after injection. Radiographs were obtained 0, 5, 10, 15, 20 and 30 min after injection and were analysed to determine the distribution and diffusion of the contrast medium. RESULTS: In 89% of injections an elongated pattern of the contrast medium was observed suggesting distribution along the neurovascular bundle. After 49% of injections a fine radiopaque line extended proximally from the contrast 'patch', and in 25% of injections a line extended distally. There was significant proximal and distal diffusion with time when sequential radiographs of each limb were compared. The greatest diffusion occurred in the first 10 min. Walking did not significantly influence the extent of either proximal or distal diffusion. CONCLUSIONS AND POTENTIAL RELEVANCE: Significant proximal diffusion occurs in the first 10 min after perineural injection in the distal aspect of the limb and should be considered when interpreting nerve blocks. Distribution of local anaesthetic solution outside the fascia surrounding the neurovascular bundle or in lymphatic vessels may explain delayed or decreased effects.

Kelvin-Helmholtz instability for relativistic fluids.

We reexamine the stability of an interface separating two nonmagnetized relativistic fluids in relative motion, showing that, in an appropriate reference frame, it is possible to find analytic solutions to the dispersion relation. Moreover, we show that the critical value of the Mach number, introduced by compressibility, is unchanged from the nonrelativistic case if we redefine the Mach number as M= [beta/ (1- beta(2) )(1/2) ] [ beta(s) / (1- beta(2)(s) )(1/2) ](-1) , where beta and beta(s) are, respectively, the speed of the fluid and the speed of sound (in units of the speed of light).

Mosaicplasty for the treatment of articular defects of the knee and ankle.

Efficacious treatment of full-thickness cartilage defects of the weightbearing surfaces is a multi-faceted challenge for the orthopaedic surgeon. Autologous osteochondral transplantation represents one solution: to bring about a hyaline or hyalinelike repair of the defected area. The current authors discuss the experimental background and their 8 years of clinical experience with the autologous osteochondral mosaicplasty. Several series of animal studies and subsequent clinical practice have confirmed the survival of the transplanted hyaline cartilage. Hyaline cartilage and fibrocartilage fill the donor sites located on the nonweightbearing surfaces and surfaces that bear less weight. Clinical scores, imaging techniques, control arthroscopies, histologic examination of biopsy samples, and cartilage stiffness measurements were used to evaluate the clinical outcomes and quality of the transplanted cartilage. According to these investigations, femoral condylar implantations have shown good to excellent results in 92%, tibial resurfacing in 88%, patellar and/or trochlear mosaicplasties in 81%, and talar procedures in 94% of patients. The Bandi score showed long-term donor site disturbances in 3% of patients. Fifty-eight of the 68 control arthroscopies had good gliding surfaces, histologically-proven survival of the transplanted hyaline cartilage, and fibrocartilage covering of the donor sites. In the entire series, there were four deep infections and 34 painful hemarthroses after surgery. A multicentric, comparative, prospective evaluation of 413 arthroscopic resurfacing procedures (mosaicplasty, Pridie drilling, abrasion arthroplasty, and microfracture cases in homogenized subgroups) showed that mosaicplasty gave a more favorable clinical outcome in the long-term followup, than the other three techniques. Intermediate-term evaluation of the femoral condylar implantations (3-6-years followup) and talar mosaicplasties (3-7-years followup) confirmed the durability of the early results. From these encouraging results from an increasingly large series and similar results from other centers, it seems that autologous osteochondral mosaicplasty may be a viable alternative treatment of localized full-thickness cartilage damage of the weightbearing surfaces of the knee and other weightbearing synovial joints.

The surgical technique and the age of the horse both influence the outcome of mosaicplasty in a cadaver equine stifle model.

Six pieces of grafts, 6.5 mm in diameter, 20 mm in length, were taken from each of 170 cadaver hindlimbs, using the cranial surface of the medial femoral trochlea for harvesting. The age of the horses varied between 4 months and 23 years. 30 limbs under the age of 12 years were selected for transplantation. Three of six grafts were transplanted into the medial femoral condyle using different combinations of tunnel depth and dilation. With ageing, a significant decline in transplantability was detected. In general, mosaicplasty cannot be recommended in horses above 11 years. Based on a previous clinical case (Bodó et al., 2000), a good surface alignment was indeed achieved with a combination of graft length drilling and dilation in most cases. However, the occasional entrapment of cartilage debris under the graft prevented perfect alignment in the present cadaver study in 27% of the grafts transplanted in this manner. Since the protrusion of grafts never exceeded 1.5 mm, we conclude that drilling 3-5 mm deeper than graft length with graft length deep dilation can avoid disadvantageous protrusion of the transplanted hyaline cartilage caps, achieving bone decompression at the same time.

Arthroscopic autologous osteochondral mosaicplasty for the treatment of subchondral cystic lesion in the medial femoral condyle in a horse.

An 11-year-old, Hungarian half-bred stallion was presented with a history of mixed left hindlimb lameness of 6 months duration. Subchondral bone cyst of the medial femoral condyle and injury of the medial meniscus were diagnosed. Osteochondral autograft transplantation (mosaic arthroplasty) was performed, taking grafts from the less weight-bearing medial border of the medial femoral trochlea of the affected limb, and transplanting them into the cyst during arthroscopy. The lameness was evaluated prior to and one year after the operation with a motion analysis system during treadmill exercise. Considerable improvement of the lameness and the clinical signs as well as successful transplantation of the grafts, and a new hard joint cartilage surface of the medial femoral condyle could be detected during follow-up arthroscopy. Osteochondral autograft transplantation seems to bee a possible alternative for treating subchondral cystic lesions of the medial femoral condyle in horses. A new technique for the surgical treatment of a subchondral cystic lesion of the medial femoral condyle in the horse is described.

Immunohistochemical evaluation of the safety of transurethral electrovaporization of the prostate and its clinical results.

Short term follow up studies on transurethral electrovaporization (TUEVP) have shown a relative low morbidity over TURP. The use of high power current has been claimed as a source of possible damage on the neuronal structures surrounding the prostate. The aims of our study were to assess longer follow up results as well as the safety of this technique. Over an 18 month mean follow up period symptom relief remained relatively stable. Postoperative dysuria was detected in a higher percentage of patients and was seen for a longer period in comparison with previous reports. Immunohistochemical staining performed using S-100 and NF monoclonal antibodies showed anatomical integrity of the prostatic neuronal fibres surrounding the vaporization edge. In conclusion, although the effectiveness and safety of TUEVP are confirmed by the present study, the occurrence of a significant rate of long-lasting postoperative irritative symptoms must be taken into account.

Circadian antidiuretic hormone variation in elderly men complaining of persistent nocturia after urinary flow obstruction removal.

Persistence of nocturia after prostatic resection in healthy patients without symptoms referred to residual bladder instability and to pathological polyuria seems to be caused by an increased urine production at night. The more accreditate mechanism involved is the relevant decreased ADH secretion pattern which occurs at night. In our study, patients with nocturnal poliuria showed significantly low plasmatic vasopressin levels compared with a control group. The aim of this study was to evaluate whether the persistence of nocturia after prostatic resection in healthy patients, without symptoms referred due to residual bladder instability and important polyuria, could be due to a decrease or a lack of increase in antidiuretic hormone (ADH) nocturnal levels following increased urine production at night. Serum ADH, atrial natriuretic peptide (ANP) and osmolality were assessed at 4-h intervals in 12 patients complaining of residual nocturia (group A) and in a control group of 13 patients who had undergone a complete resolution of nocturia after prostate ablation (group B). In the 25 patients involved in the study (mean age 65.8 years), no significant differences were observed in the two groups concerning mean age (67.5 years for group A, 64 years for group B). Mean nocturnal urine volume (1080 +/- 490 ml) in group A patients was significantly higher than in group B (500 +/- 100 ml) (p < 0.001), while no significant differences were found in diurnal diuresis. Mean plasma vasopressin levels of the 12 patients showing an increased nocturnal micturition were found to be significantly lower at all 4-h intervals when compared with the control group (p < 0.05). Individual fluctuations in serum osmolality were slight and insignificant within the normal range in all patients. The diurnal variation of plasma atrial natriuretic peptide was within the reference limits for all subjects during the 24-h period. Our results lead us to believe that residual nocturia after prostatic resection seems to be caused by an increased urine production at night due to a decreased ADH secretion pattern.

[Renal agenesis and homolateral seminal vesicle cyst. Case report]. / Agenesia renale e cisti della vescicola seminale omolaterale. Caso clinico.

The authors present a case of renal agenesis and seminal vesicle cyst in a young man, diagnosed because of several micturion disorders partially cleared up with antibiotic therapy. We discuss about the diagnosis technique which includes Ultrasound Scanner, Computed Tomography, Magnetic Resonance and pelvic arteriography. Then we describe the surgical approach and finally we discuss about embryogenesis.

[Surgical treatment of benign prostatic hypertrophy and Kelami syndrome]. / Trattamento chirurgico dell'ipertrofia prostatica benigna e sindrome di Kelami.

The authors analyze a group of 116 consecutive patients treated with transvesical prostatic adenomectomy or transurethral prostatic resection for benign prostatic hypertrophy (BPH) isolated five of them with a Kelami syndrome. These patients, aged between 53 and 70 years old, had a relevant ventral penile deflection with severe sexual impairment postoperatively. Among them only one asked for a corrective surgical procedure. Aetiology, preventive measures and therapeutic choices are discussed. Authors pointed out that this syndrome is actually underestimated considering the high number of transurethral or open surgery procedures for BPH.

[Appearance in 2 brothers of double primary neoplasms: right renal carcinoma and gastric adenocarcinoma]. / Comparsa in due fratelli di duplice neoplasia primitiva: carcinoma renale destro ed adenocarcinoma gastrico.

We report a clinical case of a double primitive tumour (right kidney clear cell carcinoma and gastric carcinoma) in two brothers. There is no history of cancer in the parents. Both patients were previously affected by gastric ulcer. No report of association between the two neoplasms was found in literature. The age of the patients (61 and 70 years) and the singleness of the kidney tumour seem to exclude the case of a familial kidney cancer. The neoplastic transformation of the gastric ulcer is instead a quite frequent report with an incidence of about 1%. Alterations of oncogenes or tumour suppressor genes shared from both neoplasm are at present still unknown. Nevertheless molecular analysis of patients' neoplastic genome could point out typical chromosome translocations/deletions or gene mutations.

[Leiomyosarcoma of the spermatic cord. Case report and revision of the literature]. / Leiomiosarcoma del funicolo spermatico. Presentazione di un caso clinico e revisione della letteratura.

A 76 year old man presented with an asymptomatic, very big left scrotal mass that was found to be a leiomyosarcoma of the spermatic cord. It was treated by radical orchiectomy with ligation of the spermatic cord at the internal inguinal ring. Convalescence was uneventful. 6 months postoperatively there was no evidence of local recurrence of the neoplasm or distant metastasis.

[Neurological evaluation of erectile impotence]. / La valutazione neurologica dell'impotenza erettile.

The diagnostic approach to neurologic evaluation of erectile impotence in neurologic subjects differs from non neurologic subjects; in particular in this last group of patients the aim of diagnosis may drive to point out or exclude an hidden or underestimated neurologic lesion. Authors report the different diagnostic approach.

Periplasmic expression of human interferon-alpha 2c in Escherichia coli results in a correctly folded molecule.

Human interferon-alpha 2c (IFN-alpha 2c) was produced in Escherichia coli under the control of the alkaline phosphatase promoter using a periplasmic expression system. Compared with other leader sequences, the heat-stable enterotoxin II leader of E. coli (STII) resulted in the highest rate of correct processing as judged by Western-blot analysis. The fermentation was designed as a batch-fed process in order to obtain a high yield of biomass. The processing rate of IFN-alpha 2c could be increased from 25% to more than 50% by shifting the fermentation pH from 7.0 to 6.7. IFN-alpha 2c extracted from the periplasm was purified by a new four-step chromatographic procedure. Whereas cytoplasmically produced IFN-alpha 2c does not have its full native structure, IFN-alpha 2c extracted from the periplasm was found to be correctly folded, as shown by c.d. spectroscopy. Peptide-map analysis in combination with m.s. revealed the correct formation of disulphide bridges. N-terminal sequence analysis showed complete removal of the leader sequence, creating the authentic N-terminus starting with cysteine.

[The use of hemorrhage occluder pins in radical pelvic urologic surgery]. / L'uso degli Hemorrage Occluder Pins nella chirurgia pelvica urologica demolitiva.

The authors present the use of "Hemorrhage Occluder Pins" in pelvic urologic radical surgery, as a means of hemostasis for the retropubic venous plexus. The application technique and the bio-technological characteristics of the occluder pin are discussed.

[Prevention of postoperative scrotal edema]. / La prevenzione dell'edema scrotale post-chirurgico.

The authors describe the technique of scrotal elevation and compression for the prevention of postoperative scrotal hematoma. This technique was used in 30 patients undergoing hydrocelectomy, spermatocelectomy or epididymectomy. There have been no untoward effects associated with this technique and this technique is applicable to a wide variety of scrotal procedures. The method described is simple, rapid to perform, comfortable for the patients and most effective to prevent postoperative complications in patients undergoing a scrotal operation.