There are a number of nonsurgical modalities used by athletes in attempts to improve performance or prevent, treat, and rehabilitate musculoskeletal injuries. A concise review of available evidence on common nonsurgical modalities used today is necessary, so that practitioners may appropriately counsel patients.A comprehensive review of relevant publications regarding cupping and blood flow restriction (BFR) from 2006 through 2019 was completed using PubMed and Google Scholar.There have been numerous investigations evaluating the efficacy of nonsurgical modalities for a myriad of musculoskeletal conditions. Cupping may be an effective option with low risk in treating nonspecific, musculoskeletal pain. Studies comparing BFR with non-BFR controls suggest that it may increase muscle strength and endurance for individuals undergoing rehabilitation or sport-specific training by mimicking the low oxygen environment during exercise.Nonsurgical modalities are low-cost treatment strategies with rates of adverse outcomes as low as 0.008% that will likely continue to increase in popularity. Despite the paucity of recent research in cupping and BFR, evidence suggests benefits with use. High-quality studies are needed to effectively evaluate these treatments, so that care providers can provide appropriate guidance based on evidence-based medicine. The sports medicine specialist enjoys a unique role in the nonsurgical and surgical care of active patients of all skill levels. However, various nonsurgical modalities represent a notable element of sports medicine and musculoskeletal care that is not traditionally taught during orthopaedic training. The increasing use of such modalities merits a review of available evidence to appropriately assess their efficacy and safety, so that clinicians are able to make informed decisions in the care of patients requesting such therapy. In part 1 of this two-part series, we analyze Kinesio taping, sports massage therapy, and acupuncture. Part 2 will discuss cupping and blood flow restriction (BFR) training.1 Unfortunately, the validity of this statement has yet to be proven. The following section will review the history, proposed mechanisms of action, and available evidence for cupping in treating musculoskeletal conditions. Given that cupping is an inexpensive and noninvasive modality, it may become an important adjunct treatment in the armamentarium of physicians treating musculoskeletal injuries.Cupping is an ancient technique that has been used to treat many disorders from musculoskeletal pain, most commonly of the back, neck, and shoulders, to medical conditions such as hypertension. Recently, there has been an increased interest in cupping, even with elite athletes undergoing treatment as evidenced by the characteristic markings seen on Michael Phelps during the 2016 Olympic Summer Games. When reporting on the use of cupping among elite athletes, the National Broadcasting Company stated that cupping is a “therapy technique that athletes use to help their muscles recover and perform at their best.”2 Although cupping has been practiced in many cultures, its true origin remains unknown. The earliest known record of cupping dates back to 1550 BC as described in the Egyptian Ebers Papyrus.4 There is also evidence that cupping, called “Al-hijamah” meaning “to restore to the original size,” was used by ancient Arabic cultures and that it was used to treat hypertension, polycythemia, headaches, and drug intoxication.5 The practice later spread throughout the world and was done by physicians, barber surgeons, and bathhouse attendants. Today, it is most commonly used by Eastern medicine practitioners. It was accepted as an official therapeutic practice in Chinese hospitals in 1999 and a 2006 Korean survey found that 93.5% of 6708 physicians used cupping in their practice.6Cupping originated over thousands of years ago and remains a notable component of complementary and alternative medicine in various parts of the world.39 Lubricants can also be used allowing movement of the cup over the skin to cover a greater surface area. The most common sites of application are the back, chest, abdomen, and buttocks.7 Cupping can be done dry or wet. In wet cupping, the patient undergoes dry cupping for several minutes at which point the suction is abated and a small skin incision is made. Disinfectants are used before making the incision and after cupping is finished to minimize risk of infection.8 Suction is then reinitiated to draw out blood that had collected underneath the skin. Finally, cupping results in characteristic skin changes that may include petechiae, erythema, edema, and ecchymosis in a circular pattern that may take days to weeks to resolve.Various cupping techniques have evolved over time; however, the use of a cup to create a vacuum of negative pressure is common to every method. Cupping is generally done using small round cups of thick glass with a rolled rim to ensure a uniform and airtight contact with skin Figures –4. Negat9 while medieval Europeans used cupping to expel evil spirits.4 In 1920, Epstein hypothesized that cupping transferred pain from one site to another thus curing the original site of pain, while a psychosomatic theory has been advocated by several research groups more recently proposing the therapeutic effect of cupping is due to a placebo effect alone.10-12 Other mechanisms of action of cupping therapy that have been hypothesized include enhancing skin circulation, loosening adhesions and lifting connective tissue, changing the biomechanical properties of skin, altering pressure pain thresholds, adjusting serum P substance levels, stimulating the peripheral nervous system, decreasing oxidative stress, and modulating the immune system.13-15 In regard to wet cupping, practitioners hypothesize that the expressed blood is rich in harmful toxins that had previously been trapped deeper in the soft tissue.17 Subadi et al. conduced a randomized control trial and found that wet cupping therapy was effective at raising the pain threshold in rats exposed to a hot plate. The authors hypothesized that this effect was attributed to expression of heat shock protein 70 (HSP70) and β-endorphins, both of which mediate pain response.18 Despite these findings, a recent extensive review of the literature by Rozenfeld and Kalichman4 concluded that that there are “no reliable scientific data clarifying the exact mechanism which can determine the therapeutic effect of cupping.”There have been many different hypotheses and theories regarding the mechanism of action of cupping. Ancient Eastern medicine believed that ailments were a result of alterations or imbalances in one's life force or “Qi” of which cupping was able to correct, similar to theories regarding acupuncture,9 In a biomechanical model, Tham et al9 illustrated that the soft tissue directly under the rim of the cup compresses while the periphery tenses. The existence of this high tensile stress eventually leads to capillary rupture resulting in the characteristic ecchymosis. Furthermore, cupping is a relaxing treatment modality not unlike massage and acupuncture, and some of its benefits may be a result of stress reduction that is not easily objectified or investigated.What is definitively known about cupping is that the negative pressure results in superficial dermal capillary dilation as a result of the high tensile stress.2 evaluated the recent research and evidence on the therapeutic efficacy of cupping concluding that there was poor clinical evidence for its use. Despite this, there is an extensive list of clinical uses that cupping has been applied to including cardiovascular disease, angina, migraine, fibromyalgia, hypertension, herpes zoster, Behcet disease, secondary amenorrhea, depression and anxiety, fatigue, metabolic syndrome, and acne vulgaris. However, its most common use is to treat musculoskeletal pain.Recently, Chen et al19 conducted a systematic review and meta-analysis on 10 RCT studies analyzing the effectiveness of cupping on back pain. The authors found that cupping therapy reduced quantitative outcomes of cervical and lower back pain in patients compared with controls, who did not receive any treatment. In addition, Lauche et al20 investigated dry cupping in an RCT where 50 patients received five cupping treatments over a 2-week period while waiting-list controls did not receive any treatment. The 36-Item Short Form Survey subscales for bodily pain and vitality were markedly improved in patients undergoing cupping, and patients subjectively reported markedly less pain. Wang et al. conducted a meta-analysis on six RCTs comparing cupping therapy with control in management of lower back pain. The authors found a notable decrease in pain and disability in the cupping group.21 Kim et al22 also conducted a systematic review and meta-analysis on 18 RCTs to analyze cupping therapy on management of general neck pain. The authors found that cupping was effective as either an add-on treatment or by itself. Cramer et al23 investigated pulsating pneumatic cupping (a method of cupping that incorporates massage) on chronic neck pain compared with standard medical care in a controlled trial with 50 patients. After 2 weeks with five cupping sessions, patients experienced notable decreases in pain intensity, pain with motion, and functional disability as measured by the neck disability index and improved physical quality of life as measured by the SF-36. Kim et al24 compared a heating pad with six sessions of wet and dry cupping in 40 patients done over a 2-week period. Cupping therapy was more effective in improving pain on the numeric pain rating scale and neck function when measured at 3 and 7 weeks of follow-up. Finally, an RCT of 60 patients in Taiwan with chronic neck and shoulder pain were randomized to no intervention or dry cupping.25 Patients who received dry cupping had a post-treatment reduction of 6.1 and 5.9 in neck and shoulder pain on a 10-point visual analog scale compared with only 0.2 and 0.6 in controls. No long-term follow-up was provided in this investigation. Despite the fact that these trials may not have been of the highest quality, that various techniques of treatment without standardization were used, and different lengths of follow-up were investigated, the results do suggest that patients may experience decreases in chronic neck, back, and shoulder pain after cupping therapy.Cupping has been investigated for the treatment of nonspecific neck, back, and shoulder pain. Castro Moura et al26 have done an RCT in Germany where 40 patients were randomized to eight sessions of dry cupping over 4 weeks or to no intervention. After 4 and 12 weeks of follow-up, there were notable differences found in the Western Ontario and McMaster Universities Osteoarthritis global score, pain intensity on the visual analog scale, and physical component of the SF-36 scale. This led the authors to conclude that pulsatile cupping relieved symptoms of knee OA compared with no intervention. Khan et al27 also have done a randomized controlled trial where cupping was found to be as effective to oral acetaminophen in regard to analgesia and decreasing edema related to OA in short-term follow-up.Cupping has also been investigated for knee pain associated with osteoarthritis (OA). Teut et al28 In addition, in an RCT by Michalsen et al,29 patients receiving wet cupping therapy experienced greater improvement in severity of CTS after a single treatment session compared with the control group.Finally, cupping therapy has been found to be effective in the management of carpal tunnel syndrome (CTS). In an RCT of 56 hands with CTS, individuals treated with cupping therapy and physiotherapy  experienced notable improvement in severity of CTS and distal sensory latency compared with the control group receiving only physiotherapy.30 recently studied the use of alternative medicine in the treatment of sports injuries among national Korean volleyball players. In a survey distributed to six team doctors who treated a total of 166 injuries over a single season, cupping was actually one of the least frequently used traditional treatments (7.9%), while acupuncture was the most commonly used (40.4%). However, no data on treatment efficacy were reported.Unfortunately, there are limited data on the use of cupping in treating athletes and its plausible effects on performance. One investigation done by Yang et al31-33 In an RCT conducted by Kim et al,24 four of 40 participants (10%) experienced adverse events associated with cupping . Conversely, a cross-sectional survey of residents in Korea by Song et al34 found that none of the 45 respondents experienced adverse events associated with cupping therapy. Studies suggest that cupping should not be done greater than 20 minutes to avoid blistering.35There is variation in the number of adverse events documented related to cupping. These include abscess formation, postinflammatory hyperpigmentation, anemia requiring transfusion, keloids, burns, and blistering.There has recently been an increasing interest in cupping and wet cupping among elite athletes and the general cohort. Despite the growing prevalence of cupping therapy, there is a paucity of research, guidance, and regulation particularly regarding its efficacy for improving athletic performance. The available literature may support the use of cupping in treating musculoskeletal pain associated with nonspecific neck, back, and shoulder pain, but there are a lack of recent studies, and trials in the literature are not of high quality. The authors agree with multiple investigations reviewing the use of cupping, in that further investigations are necessary that standardize the therapy used, increase the study cohort, provide sufficient long-term observation, and look at objective end points. Nevertheless, given the available data, the risks associated with properly done dry cupping are low enough that physicians can feel comfortable recommending its use among interested athletes experiencing shoulder, neck, or back pain.36 More recently, BFR training has entered mainstream media because of its prevalent use by professional athletes across a range of sports. The basic premise of BFR is the use of low-load resistance exercise combined with moderate BFR to increase muscle mass and strength, and thereby, enhance sport-specific performance.38 Typically, a flexible cuff is placed around either the proximal thigh or arm and pressurized to an appropriate level to maintain arterial inflow to the muscle group while occluding venous return during exercise.39 The suggested pressure recommendation to achieve maximum anabolic response is approximately 50% arterial occlusion.40 Thus, the objective of BFR training is to simulate and receive the benefits of high-intensity resistance exercise while doing low-intensity resistance exercise.41 As such, BFR training therapy holds tremendous potential for maximizing athlete strength, endurance, sport-specific performance, and rehabilitation from injury.BFR training (or Kaatsu training) was initially developed in Japan in the mid-1990s.42 Third, active skeletal muscle produces metabolites such as lactic acid, hydrogen ions, and various other catabolites that invoke multiple anabolic signal transduction pathways leading to skeletal muscle hypertrophy and hyperplasia.43 Finally, the low oxygen tension in exercising skeletal muscle hastens the recruitment of fast-glycolytic muscle fibers that have high-force–generating capacity.44Under normal exercise conditions, the increased oxygen demand of skeletal muscle is met by a variety of mechanisms. First, a greater percentage of the cardiac output is routed to the exercising muscle. Second, there is vasodilation of the vascular network of the active muscle with simultaneous vasoconstriction of peripheral parasympathetic vascular beds. This constrictive device selectively restricts venous outflow, thereby leading to accumulation of the metabolites, such as lactic acid, in the surrounding capillary network.47 Depending on the objective (rehabilitation versus athletic training), a cuff can be placed either unilaterally or bilaterally.48 In effect, the metabolic environment created within the muscle is similar to what the muscle experiences when undergoing high-resistance exercise. The end result is activation of multiple signaling pathways leading to skeletal muscle hypertrophy.BFR training attempts to exploit such physiological adaptations of exercise. High-resistance exercise is simulated by extrinsically reducing blood flow to active skeletal muscles during periods of low-resistance exercise . A pressurized inflatable cuff or elastic band is placed immediately proximal to the exercising muscle.49 evaluating laboratory markers between an experimental cohort doing bilateral knee extension exercises with BFR against a matched-control cohort doing bilateral knee extension exercises with unrestricted resistance. The BFR cohort demonstrated markedly higher levels of lactate and growth hormone; in addition, there was no difference between the cohorts with respect to degradative catabolic markers such as creatine kinase (a measure of muscle breakdown) or lipid peroxide (a measure of oxidative stress).The anabolic potential of BFR training was supported in a study by Takarada et al51 For example, the metabolic response to BFR training varies between endurance and sprint runners.52 BFR training resulted in higher levels of physiologic stress as evidenced by lower phosphocreatine and intramuscular pH levels in endurance runners compared with sprint runners. These differences are likely the result of pre-existing differences in muscular adaptation that occur during a career of endurance versus sprint running. Quite possibly, sprint runners routinely do under conditions of low intramuscular oxygen tension and thus are not challenged by the metabolic environment created during BFR training. Regardless, the physician and training staff must be aware that different athletes across different sports may have different physiologic responses and adaptations to BFR training.There can be variability in the physiologic response to BFR training among athletes similar to the variability seen in the physiologic adaptation to high-resistance training seen among athletes.53-55 Takarada et al examined the effects of combining resistance training with BFR in elite rugby players in Japan.33 Study participants have done 8 weeks of low-load resistance training consisting of bilateral knee extensions with or without BFR. The group that did exercises with BFR demonstrated increased muscle endurance compared with the group without BFR. Furthermore, the group that did exercises with BFR demonstrated an increased cross-sectional muscle area at the end of the study compared with the beginning of the study. Even seasoned athletes have achieved benefits from incorporation of BFR training. Yamanaka et al55 had Division IA football players with at least 5 years of resistance training experience do low-load training with and without BFR. The athletes who have done low-load resistance training with BFR demonstrated a markedly greater increase in their 1 repetition maximum for bench press and squats than the group that did not incorporate BFR; furthermore, the football players using BFR demonstrated a markedly greater increase in chest girth.Several studies have demonstrated increased muscle strength, endurance, and cross-sectional area with low-resistance BFR training.56 conducted a systematic review and meta-analysis comparing low-load BFR with high-load BFR in changes to muscle strength and muscle mass. The authors identified 12 studies analyzing muscle strength outcomes between low-load and high-load BFR and 10 studies analyzing muscle mass outcomes between low-load and high-load BFR. The authors found that high-load BFR was superior to low-load BFR in increasing muscle strength, but was comparable with respect to increases in muscle mass.Lixandrão et al57-59 Luebbers et al57 had American collegiate football players train 4 days each week for 7 weeks in one of four groups: (1) traditional high-load training, (2) traditional high-load training supplemented with low-load training, (3) traditional high-load training supplemented with low-load BFR training, and (4) modified traditional training supplemented with low-load BFR training. The third group doing traditional high-load training supplemented with low-load BFR training demonstrated the largest increase in squatting 1-repetition maximum. In another similar study evaluating recreational male athletes, high-load unrestricted resistance exercise combined with low-load BFR training was the exercise regimen that led to the greatest increase in maximal isometric elbow extension.58Multiple studies comparing different training programs have shown low-load BFR resistance training combined with traditional high-load resistance training leads to maximal strength gains.60 Similarly, athletes across a variety of sports who underwent low-load BFR training have shown improved performance in 505 agility and 20-m shuttle-run tests.53The enhanced muscular adaptations accrued through BFR training have been shown to increase performance in sports-specific tests. Collegiate track and field athletes using low-load BFR training have shown improved 10-meter acceleration and 30-meter sprint times.62 In an RCT by Cancio et al,63 BFR training after closed treatment of distal radius fracture reduced pain with activity and increased self-perceived function compared with the control groups. Hughes et al64 conducted a systematic review and meta-analysis on patients with musculoskeletal conditions undergoing low-load BFR training for rehabilitation. They concluded low-load BFR increased strength, but was less effective than heavy-load training. Similarly, Centner et al65 also conducted a systematic review and meta-analysis of 11 studies on BFR training in older individuals and found that low-load BFR training had similar effects to non-BFR heavy-load training on muscle hypertrophy, but had lower increases in strength. Loenneke et al45 have proposed a progressive model for incorporating BFR training from the early phases of rehabilitation through to the resumption of high-load sport-specific training. This model comprised four sequential phases: (1) BFR alone during periods of immobilization, (2) BFR during low-work rate walking, (3) BFR during low-load resistance exercise, and (4) low-load BFR training combined with normal high-load training.Finally, low-load BFR training can be beneficial for athletes during rehabilitation from injury. In patients recovering from anterior cruciate ligament reconstruction, BFR training can attenuate muscle atrophy and enhance muscle development.39 in a review of available peer-reviewed English language studies investigating BFR training in the athletic cohort were only able to find 11 investigations that met their inclusion criteria. Studies also demonstrate variability in the BFR stimulus. For example, some investigators use inflatable cuffs to achieve strict control of the BFR stimulus while other studies apply elastic wraps to achieve BFR. The use of elastic wraps for BFR has been demonstrated to provide safe, effective, and valid occlusive stimulus for BFR training.66 However, the exact pressure applied to limbs is not known when using an elastic wrap. Although strict control of pressures is possible with inflatable cuffs, there is variability among studies in pressures calibrated, and studies do not always report pressures applied. The effect that different BFR pressures have on muscular adaptations is not completely understood.At present, the widespread adoption of low-load BFR training is hindered by the limited number of high-quality published studies and variability in BFR utilization. Scott et al57 However, these measurements can be imprecise. Furthermore, during the course of a variable training regimen over a period of days to months, the distribution and composition of tissues can change, thus confounding measurements.55The literature regarding BFR training in athletes also demonstrates variability in clinical outcomes reported and methodological variability in assessment of outcomes. For example, studies may report limb and/or torso girth measurements as a proxy for muscle hypertrophy.68 In addition, low-load BFR training does not necessarily increase the strength of the entire musculotendinous unit.The current literature has shown that low-load BFR resistance training can increase muscle strength, endurance, and cross-sectional area. However, low-load BFR resistance training produces lower levels of muscle recruitment than high-load resistance training with and without BFR.69 In addition, nerve compression from excessively high cuff pressure can result in transient numbness and tingling.70A large increase in muscle strength with no corresponding increase in surrounding connective tissue strength may lead to tendon (or connective tissue) injury, especially when athletes attempt to lift heavy loads that can be accommodated by muscles but not tolerated by connective tissues.33 A case report describes a male ice hockey player recovering from injury with BFR training and being diagnosed with rhabdomyolysis.71 A survey of 105 training facilities in Japan reported rhabdomyolysis occurring in 0.008% of BFR trainees.72Finally, as with any exercise regimen, there is potential risk of injury with BFR training. Only sparse injuries have been reported in the literature in the setting of BFR training.Although the body of recent evidence is limited, the current literature suggests that low-load BFR training can increase muscle strength, endurance, cross-sectional area, and potentially sport-specific performance. In addition, low-load BFR training holds promise for enhanced recovery from injury. Finally, for the elite athlete, maximal strength benefits may be achieved by supplementing traditional high-load training with low-load BFR training.


A 63-year-old female presented to the emergency department complaining of cough, neck swelling, dysphagia, and dysphonia for two days, with a past medical history of atrial fibrillation managed with warfarin. Investigations revealed a supratherapeutic international normalised ratio (greater than 10). Imaging and endoscopic examination showed an extensive retropharyngeal hematoma with significant mass effect on the airway.A rare but potentially fatal complication of warfarin anticoagulation is upper airway hematoma, with violent coughing described as an inciting cause. Signs of airway compromise necessitate specialist consultation and definitive airway management, while mild cases without airway concerns can be managed conservatively with medical anticoagulation reversal. A 63-year-old female presented to the emergency department with a two-day history of cough, neck swelling, dysphagia, and dysphonia. She was taking warfarin for atrial fibrillation. Vital signs were normal, and she was not in respiratory distress. Further examination revealed hoarseness and multiple ecchymoses over the anterior aspect of her neck , as wellImmediate anesthesiology and otorhinolaryngology consultations were requested. Non-contrast computed tomography and endoscopic examination revealed a hematoma extending from the retropharyngeal space of the suprahyoid neck, down into the infrahyoid neck to the level of the thyroid cartilage, causing significant mass effect on the airway and right vocal cord .The patient’s international normalised ratio (INR) was supratherapeutic, measuring greater than 10. She was managed conservatively, with 10 mg of vitamin K, 3000 units of prothrombin complex concentrate (PCC), as well as 10 milligrams of dexamethasone, and observed in the intensive care unit (ICU). She was transferred to the surgical observation unit the following day, and discharged home six days later.Upper airway hematomas are a potentially fatal albeit rare complication of warfarin anticoagulation, with violent coughing described as an inciting cause.1CPC-EM CapsuleWhat do we already know about this clinical entity?Upper airway hematoma is a rare but potentially fatal complication of warfarin therapy.Signs of airway compromise necessitate prompt airway management.What is the major impact of the image(s)?Initial clinical appearance of unexplained ecchymoses in anticoagulated patients should increase the suspicion of a hematoma in the airway.How might this improve emergency medicine practice?Rapid reversal of anticoagulation with vitamin K and prothrombin complex concentrate may prevent the need for airway intervention.


To report the effectiveness of an intraoperative posterior chamber irrigation technique to enhance vitreous cavity support during postvitrectomy phacoemulsification cataract surgery.  The irrigation technique was performed during phacoemulsification cataract surgery on 10 postvitrectomy eyes (9 patients) with moderate or hard density cataracts and with low vitreous cavity support. A cohesive viscoelastic tamponade was applied to pressurize the anterior chamber to start the procedure. The vitreous cavity was then irrigated using a 26-gauge flushing cannula injecting balanced salt solution under the iris through the zonules, until the vitreous cavity pressure balanced and exceeded the anterior chamber pressure and viscoelastic flowed out from the corneal incision. Intraoperative performance with the irrigation technique, postoperative visual acuity, and anatomy, and complications were retrospectively evaluated.  The irrigation procedure instantly enhanced posterior segment pressure before capsulorhexis in 4 eyes, before phacoemulsification in 4 eyes, after phacoemulsification in 2 eyes, before intraocular lens implantation in 6 eyes, and after implantation in 3 eyes. Phacoemulsification cataract surgery was facilitated by the irrigation technique, with a stabilized anterior chamber and robust vitreous cavity support. No complications occurred intraoperatively and postoperatively. At a 3-month follow-up, favorable visual and anatomic outcomes were achieved in all eyes.  The irrigation technique balanced the pressure of anterior and posterior segments. Thus, vitreous cavity support and anterior chamber depth were well stabilized during phacoemulsification cataract surgery in postvitrectomy eyes. Cataract is the most common complication of pars plana vitrectomy (PPV) used extensively to treat various posterior segment pathologies , 2. PhacCurrent anterior segment strategies for a safer cataract surgery in postvitrectomy eyes involve accurate incisions, improved fluidics, and utilization of ophthalmic viscosurgical devices (viscoelastic) , 15. TheHerein, we described an intraoperative posterior chamber irrigation technique in which the anterior chamber was pressurized with viscoelastic and the vitreous cavity was irrigated across the zonules with balanced salt solution. We evaluated the effectiveness of the technique to restore vitreous cavity volume throughout phacoemulsification cataract surgery after PPV.The posterior chamber irrigation technique . Phacoemulsification surgeries were performed under peribulbar anesthesia. Briefly, routine surgical steps were as follows: A 3.0 mm clear corneal incision and a 1.0 mm side port were made. A 5.0 to 5.5 mm continuous curvilinear capsulorhexis was performed after cohesive viscoelastic  was injected into the anterior chamber. Phacoemulsification using a “stop-and-chop” technique was performed with a 30-degree phaco tip  in a continuous mode and torsional phaco power between 40 and 60% combined with a longitudinal phaco power between 0 and 30% depending on the density of the nucleus. Irrigation bottle height was between 65 and 80 cm HThe posterior chamber irrigation procedure was applied before some major steps of cataract surgery when insufficient vitreous support was noted. Indications of insufficient vitreous support included soft scleral wall, excessive deep anterior chamber, and prominent posterior retropulsion of the iris-lens diaphragm after anterior chamber being inflated with viscoelastic or balanced salt solution , 13, 16.Anterior-to-posterior fluid flow during the irrigation procedure was traced with 2.5 mg/mL indocyanine-green  irrigation in one cataractous eye, which subsequently required additional vitrectomy and staining of the epiretinal membrane to treat recurrent retinal detachment . In the All the patients were routinely examined one day, one week, one month, and three months after surgery. Main outcomes included intraoperative performance with the irrigation technique, postoperative best-corrected Snellen visual acuity, anatomy, and complications. .The posterior chamber irrigation technique was performed on ten eyes from nine patients during phacoemulsification cataract surgery. Nine eyes with an attached retina received phacoemulsification and in-the-bag IOL implantation, and two eyes of these eyes received vitrectorhexis to remove posterior capsule plaque after IOL implantation. One eye with recurrent retinal detachment had phacoemulsification followed by a PPV with a silicone oil tamponade.During cataract surgery, the irrigation procedure was performed before capsulorhexis in four eyes , before Reasonable visual improvement and good anatomic outcomes were observed in all eyes at the three-month follow-up visit. IOLs were centered in the capsular bag, and the retinopathy was stable and intraocular pressure normal.Anterior segment methods have been established for a safer phacoemulsification cataract surgery after PPV , 14, 16,The posterior chamber irrigation procedure via an anterior segment approach quickly restores vitreous cavity volume when pressure on the anterior and posterior sides is balanced. The underlying principles are as follows: (1) the amount of viscoelastic tamponade in the anterior chamber sets the volume and pressure for anterior-to-posterior irrigation to the vitreous cavity; (2) the pathway of transzonular fluid flow depends on the pressure disparity between the anterior chamber and the posterior segment. Thus, when the vitreous cavity volume is inadequate, the irrigation distributes more posteriorly than anteriorly. Once the vitreous pressure reaches the anterior chamber pressure, reflux of fluid flushes out the viscoelastic. In our experience, a slightly deep anterior chamber with viscoelastic tamponade may be optimal. Too much viscoelastic may cause a pressurized eye and inadequate vitreous volume to accommodate liquid flow-in. Conversely, a shallow anterior chamber with less viscoelastic allows excessive fluid flow into the vitreous cavity, which may induce hypertension of posterior segment, anterior elevation of iris-lens diaphragm, and prolapse of iris, mimicking the acute fluid misdirection syndrome . DispersThe irrigation technique provided intraoperative stability of the anterior chamber with no complications. Without adequate vitreous support, rupture of the posterior capsule and nucleus drop can occur at different times . Thus, sIris irritation and damage to zonular fibers were concerns using our technique; however, such problems did not occur in this case series. The technique is not designed for eyes with extensive zonular deficiency, as fluid rush may lead to further zonular dehiscence or fluid misdirection. To minimize the risk of damage during irrigation, we recommend an adequate viscoelastic tamponade, gentle irrigation dispersion under the iris, and avoidance of injection toward the region with weak zonules.A limitation of the current study is the application of the procedure to a limited number of cases that needs to be evaluated in future studies on more cases with different clinical scenarios. Real-time intraocular pressure during cataract surgery was not measured using a tonometer which could be beneficial in future studies. Low vitreous support was reflected in real time by a soft scleral wall and excessive bowing of the iris with a deep anterior chamber. In our experience, these symptoms are reliable in judging the timing of using the irrigation procedure during cataract surgery.In conclusion, the intraoperative posterior chamber irrigation effectively restored vitreous cavity support in cataract surgeries after PPV. The simple maneuver allowed management of fluid flow for a safe phacoemulsification cataract surgery in our small cohort of patients. The safety of our technique in different clinical scenarios still needs to be confirmed in larger studies. Nevertheless, this anterior segment technique is an alternative to the technique of pars plana infusion.


Excess post-exercise oxygen consumption (EPOC) of children could indicate the potential of an exercise therapy to treat or prevent obesity. However, EPOC as a result of playing active video games (AVG) has been poorly investigated. Therefore, we aimed to investigate the rapid component of EPOC of children with healthy weight and overweight/obesity (according to their BMI percentile) after playing AVGs that feature predominately upper body (UB) and whole-body (WB) movement.2) and carbon dioxide production (VCO2) were recorded during exercise and post-exercise recovery period. For the rapid component of EPOC in each AVG session, measurements were recorded every 15 s for 5-min of post-exercise recovery. The rate of perceived exertion (RPE) was also measured immediately before and after each AVG play.Twenty-one children with healthy weight (BMI percentile < 85%) and with overweight/obesity (BMI percentile ≥ 85%) randomly underwent two 10-min AVG sessions (UB and WB). The heart rate (HR), minute ventilation (VE), oxygen consumption  during post-exercise recovery. RPE, HR, and HR% were not different between the game sessions and weight groups. Children with overweight/obesity showed a higher absolute VO2 during EPOC than healthy-weight children in both game sessions, but relative VO2 was higher in healthy-weight children during EPOC. No differences were observed for EPOC between UB and WB sessions.Children with overweight/obesity had a higher average of absolute VE, VO2 when controlling for body mass. UB and WB AVGs induced a similar EPOC among children with healthy weight and overweight/obesity. As UB and WB AVGs induce the rapid component of EPOC in children regardless their weight status, AVGs could be used as an exercise method to treat and prevent child obesity.Children with overweight/obesity had a greater EPOC than healthy-weight children after AVG sessions in terms of absolute oxygen values, whereas healthy-weight children have higher EPOC considering relative VO In the United States, 18.5% of children and adolescents are obese . MoreoveOne of the most popular strategies used to treat or prevent obesity is the reduction of sedentary behavior and increase physical activity levels . In the The literature on new AVG technologies displays a considerable heterogeneity in games, which is likely due to new releases of cutting-edge technologies every year . For insThe heterogeneity of AVGs presents an advantage of this method, which allows people with a diverse range of mobility to play/exercise. People with extremity disabilities can play AVGs targeting the upper-body (UB) or the lower-body (LB) based on their particular limitations. Other people who need to avoid the risk of acute peripheral fatigue or muscular imbalances in the long term can play whole-body (WB) AVGs, which involve more body segments . ResearcWhile energy expenditure during an activity, such as an AVG, give us information regarding the possible impact that play could have on preventing or treating obesity, measures during the post-exercise recovery period also presents an opportunity to investigate caloric expenditure and substrate oxidation rate. After exercise, the body shifts from using carbohydrate (CHO) to fat as its main fuel substrate . This chEPOC is influenced by many factors, including fitness level, nutritional status, physical activity, and level of adiposity , 18, 19.Therefore, we aimed to investigate the rapid component of oxygen consumption and substrate utilization of children with healthy weight and overweight/obesity after playing UB and WB AVGs. Our initial hypotheses were that WB AVG would elicit a greater EPOC than UB AVG, and that overweight/obese children would produce a greater absolute EPOC than children with healthy weight regardless of the type of game.Our cross-sectional study was approved by the institutional review board at the Northeastern University. All children participants signed a written informed assent and their parents or guardians, a consent for the children’s participation. All protocols were carried out in accordance with relevant guidelines and regulations.We recruited participants from a community in the Greater Boston area through web advertisements and flyers. We included boys and girls from all ethnic backgrounds. Our inclusion criteria were: aged between 8 to 12 years; free of attentional disorders or physical disability; free of any medications that could affect the central nervous system; had not previously played the AVG we intended to use in our study. Of our initial pool of 421 survey respondents, 82 children were eligible; from the latter group, we recruited 42 children. We excluded 10 participants from our final analysis due to invalid metabolic data . We have reported the details of our selection process in a previous paper , and UB [383 (95% CI: 344–416) vs. 316 (95% CI: 301–330)] game session. See Fig. 2 values, children with overweight/obesity showed a declining oxygen uptake, ending the with an average value below resting VO2 after UB  WB AVG would elicit a higher EPOC than UB AVG; 2) children with overweight/obesity have a higher EPOC than healthy-weight children regardless of the type of game. Our main findings did not support our first hypothesis as there was no significant difference between the two types of AVGs. Our second hypothesis was supported: children with overweight/obesity showed a greater oxygen consumption than healthy-weight children after both WB and UB AVG sessions. However, after controlling oxygen uptake by body weight, the findings were reversed: EPOC was higher in healthy-weight children. Additionally, we did not find differences between the two game types concerning EPOC in healthy weight and children with overweight/obesity.2peak) suggested an EPOC pattern similar to conventional cycling in the ergometer (lower body only), with a greater EPOC magnitude at higher intensities [We are the first to investigate the effect of the type of AVG over EPOC. When previous researchers compared different exercise modes, although WB or LB exercises require the involvement of more muscle mass , there iensities .Although the type of game did not affect EPOC, we did find differences between children with healthy weight and overweight/obesity, which corroborated a previous study on 10 children with obesity and 10 non-obese controls using a running/walking exercise protocol that only the obesity group showed higher RER values after exercise . Convers2max). Further investigation is needed to identify methods to equalize exercise intensity during active video games. From a practical perspective, AVGs could be used as a strategy to increase the physical activity necessary to elicit EPOC regardless of the game type (UB or WB) in both groups, with and without overweight/obesity. AVGs may thus be more efficient in producing weight loss in children with overweight/obesity.The hours after exercise represent the slow component of EPOC. Since we could not assess this part of EPOC, this may represent a limitation on our results. However, previous AVG research indicates that the exercise intensities necessary to elicit prolonged EPOC are unlikely to be tolerated by non-athletic individuals  and possBoth UB and WB AVG induce the rapid component of EPOC in children with healthy weight and overweight/obesity. Additionally, children with overweight/obesity showed a greater EPOC than healthy-weight children after AVG sessions when considering absolute oxygen values. When oxygen uptake is relative to their body weight, healthy-weight children showed a higher EPOC than children with overweight or obesity. Our results show the potential of AVGs to elicit higher fuel utilization at rest after a play session, offering potential for child obesity prevention and intervention. Future researchers could investigate the slow component of EPOC after AVG and factors related to the potential mechanisms of the change in substrate utilization during EPOC, such as insulin resistance.


Cancer biomarkers have transformed current practices in the oncology clinic. Continued discovery and validation are crucial for improving early diagnosis, risk stratification, and monitoring patient response to treatment. Profiling of the tumour genome and transcriptome are now established tools for the discovery of novel biomarkers, but alterations in proteome expression are more likely to reflect changes in tumour pathophysiology. In the past, clinical diagnostics have strongly relied on antibody-based detection strategies, but these methods carry certain limitations. Mass spectrometry (MS) is a powerful method that enables increasingly comprehensive insights into changes of the proteome to advance personalized medicine. In this review, recent improvements in MS-based clinical proteomics are highlighted with a focus on oncology. We will provide a detailed overview of clinically relevant samples types, as well as, consideration for sample preparation methods, protein quantitation strategies, MS configurations, and data analysis pipelines currently available to researchers. Critical consideration of each step is necessary to address the pressing clinical questions that advance cancer patient diagnosis and prognosis. While the majority of studies focus on the discovery of clinically-relevant biomarkers, there is a growing demand for rigorous biomarker validation. These studies focus on high-throughput targeted MS assays and multi-centre studies with standardized protocols. Additionally, improvements in MS sensitivity are opening the door to new classes of tumour-specific proteoforms including post-translational modifications and variants originating from genomic aberrations. Overlaying proteomic data to complement genomic and transcriptomic datasets forges the growing field of proteogenomics, which shows great potential to improve our understanding of cancer biology. Overall, these advancements not only solidify MS-based clinical proteomics’ integral position in cancer research, but also accelerate the shift towards becoming a regular component of routine analysis and clinical practice. Cancer is the second leading cause of death and poses a major problem to healthcare systems worldwide. The prevalence of cancer remains stable with an estimated 1.7 million new cases, resulting in 600,000 new deaths, in 2018 in the United States alone [Other fields of clinical research attempt to elucidate molecular differences between cancer cases and healthy controls or different stages of cancers as the disease progresses. These include genomics and transcriptomics that have identified numerous cancer-driving genes. While these omics datasets have demonstrated the ability to compare and contrast different clinical cancer groups, one limitation is that these changes do not necessarily directly translate to our understanding of disease biology. On the other hand, proteins are the biomolecules that directly carry out most biological processes suggesting they are ideal predictors of disease progression . AdditioMultiple studies have shown that globally mRNA expression is positively, but weakly, correlated with protein expression –6. This There currently exists a strong bias towards antibody-based techniques for the detection of clinically-relevant proteins. ELISA is commonly used to quantify protein biomarkers in a variety of biofluids, with ongoing improvements, such as Prostate-specific antigen (PSA) in the blood of suspected prostate cancer (PCa) patients as low as one hundred picograms per millilitre . ImmunohIn this review, we highlight relevant literature related to MS-based clinical proteomics with a specific focus on cancer research. We specifically focus on clinical sample types, sample preparation techniques, MS configurations and protein quantitation strategies. To limit this review to a more manageable scope we further describe notable studies that specifically investigate the proteomes of cancer tissues and bodily fluids. While we attempted to be as complete and inclusive as possible, we apologize to authors whose papers were not cited as part of this review.A wide array of sample types has been analyzed by clinical proteomics. First and foremost, larger cohorts of primary patient materials in the form of tissue samples are becoming increasingly feasible, due to improvements in biobanking and proteomics technologies. As a result, the direct proteomic investigation of clinical tissues is becoming increasingly popular. Preservation of the tissue’s proteome dynamics is critical from time of surgical resection to the protein digestion stage, and there are a few methods of doing so: fresh frozen (FF), formalin-fixed paraffin embedded (FFPE), and optimal cutting temperature embedded (OCT). One important caveat for consideration in clinical tissue-based proteomics is that surgical procedures could possibly take hours from the time a patient is admitted to the operating room to the point of sample retrieval and preservation. How this affects a tissue proteome is currently poorly understood. More rapid procedures such as needle biopsies have the potential to overcome some of these complications but provide significantly lower amounts of tissue for proteome analysis. While FF is the preservation method of choice from a proteome coverage perspective, FFPE tissues have been banked for decades, providing extensive clinical follow-up and an invaluable resource for clinical proteomics. Previously, cross-linking-based modifications produced insufficient proteomic coverage for global proteomic studies of FFPE tissues. Improvements in sample preparation have led to more efficient de-crosslinking of fixed proteins, and as a result, greater protein availability for digestion. Tissue samples can be further prepared by laser-capture microdissection (LCM) to add an element of spatial resolution. This allows different regions of the same tissue sample to be compared, whether normal adjacent or tumorous.While tissue samples have the potential to provide novel biological insights, many clinical proteomics studies have aimed to discover novel biomarkers, ideally in clinical samples that are obtainable in a non-invasive or minimally invasive manner (i.e. liquid biopsies). The most commonly analyzed biofluids are blood  and urine, but other biofluids that have been analyzed by proteomics include post-digital rectal examination urine , expressClinical sample cohorts of the past were often underpowered due to biobanking limitations . As a result, various model systems have been developed to facilitate the discovery of new biomarkers or aid in characterizing proteins of interest, as in Fig. Sample preparation plays an important role in the proteomic characterization of clinical samples and rigorous standard operating procedures need to be established in order to get relevant information on the complex biological processes that lead to cancer progression. There is no universal protocol for proteomic sample preparation, but rather the selected strategy should be optimized/selected based on the proteomic complexity, the available quantity of sample and the goal of the study. The first step in sample preparation for MS includes lysis and extraction of proteins from the clinical samples. This includes extraction reagents such as different organic solvents and detergents followed by tissue disruption techniques such as freeze–thaw cycles, sonication or mechanical disruption to maximise the protein extraction and solubilisation. Organic solvents solubilise and denature proteins and can be easily removed by evaporation using lyophilization. TFE  based lysis and extraction on nano-scale (30 µg) and macro-scale (> 100 µg) input materials gave comparable protein detection rates relative to the traditional detergent-based methods . While tDenaturants (urea and guanidine HCl), ionic detergents , and non-ionic detergents  act to efficiently lyse cells and solubilise protein complexes, especially membrane proteins. The disadvantage of detergent use is their difficult removal from samples for downstream MS applications that could lead to peptide ion suppression. Detergents also tend to deposit in the electrospray emitters and liquid chromatography lines, C18 chromatography columns, and in the MS instrument front-end causing added maintenance. Many MS-compatible, commercially available detergents have been reported and widely used including Rapigest (Waters), ProteaseMax (Promega), Invitrosol (Thermo). These detergents degrade with the addition of heat or acidic pH conditions; hence reducing problems described above. Recently different protein purification techniques have been developed with increased efficiency, reduce protein digestion time requirements and minimized sample losses as described below.The anionic surfactant sodium dodecyl sulfate (SDS) is an excellent agent to solubilise proteins but possess limited compatibility with MS applications. The removal of SDS from the peptide sample has proven to be a major barrier with conventional methods. Recently developed sample preparation methods focus on using SDS and other denaturants as the solubilization agent and their removal through various membrane-based protein capture techniques. One of these developed methods is filter aided sample preparation (FASP) first described by Manza et al.  and furtClinical proteomics studies consist of increasingly large cohorts which require efficient and timely sample preparation; which has been facilitated by the development of 96-plate format techniques. The MStern method was developed to overcome the problem of slow liquid transfer through nitrocellulose membrane. MStern uses hydrophobic PVDF membranes with significantly larger pore sizes allowing for improved liquid transfer and more efficient protein adsorption relative to nitrocellulose . A vacuuAnother recently developed membrane-based method that uses a similar principle as FASP is called suspension trapping (S-trap) . S-Trap Clinical tissue samples are sometimes challenging to process due to their small size, particularly LCM samples. These samples require efficient sample processing techniques to ensure limited sample losses and maximal extraction of the proteins to maximize proteome coverage. The solid-phase-enhanced sample preparation (SP3) method was developed with these limitations in mind. Originally described by Hughes et al.  the methThe in-StageTip (iST) method developed by Kulak et al.  in 2014 The work by Sielaff et al.  showed aThe field of clinical proteomics has seen advancements at the sample preparation stages, as well as in MS technology. MS-based proteomics’ emergence can be attributed to the development of soft ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Further advancements in front end technologies have propelled clinical proteomics to further depths of the human proteome. ESI continues to rely on well-established reversed-phase nano-LC technologies, or combined with capillary electrophoresis , and is The majority of discovery-based clinical proteomic studies continue to depend on data-dependent acquisition (DDA) to identify potential biomarkers or gain biological insights. This approach has some benefits including well established instrument operation, data analysis and processing pipelines. Additionally, DDA includes the option of label-dependent quantitation and associated multiplexing. On the downside, DDA is hampered by low inter-sample reproducibility of peptide detection due to random sampling, thus creating a “missing value” problem. While the premise of DDA has remained largely unchanged, improvements in the instrument’s efficiency have stemmed from the development of the BoxCar data-acquisition method . Unlike Due to the shortcomings of DDA, the field of clinical proteomics is observing a shift towards data-independent acquisition (DIA), a method that was originally described by Purvine et al.  and furtMS instruments continue to show versatility with various peptide fragmentation and detection configurations. Collision-induced dissociation (CID) was first introduced in 1981 and is one the most fundamental fragmentation techniques in proteomics . The ionElectron-transfer dissociation (ETD) is an ion–ion collision fragmentation-based method where cations (peptides or proteins) collide with charged radical reagent anions . ETD is There is a large number of global and targeted protein quantitation approaches available, each with their unique sets of advantages and disadvantages. These can be categorized as relative or absolute quantitation, of which the former can be further divided into label-dependent and label-free techniques.Relative quantitation often depends on the use of stable-isotopic labels that result in covalently derivatized peptides. Past methods include metabolic and chemical labelling, such as dimethylation and Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) which rely on MS1-level quantitation, but they are most applicable to cell line-based studies , 76. AddMore recent labelling strategies, such as Tandem mass tag (TMT) and isobaric tag for relative and absolute quantitation (iTRAQ), have gained popularity as these techniques apply isobaric tags providing MS2-level relative quantitation while boosting the MS1 signal intensity to improve chances of peptide detection , 80. A pThe use of stable-isotopic labels does create added expenses in clinical proteomic studies. As an alternative, recent advances in data computation have given rise to powerful label-free proteomic techniques, with sample peptides often separated over long LC gradients. Relative protein abundances are derived from MS1-level peptide peak integration using software like MaxQuant , ProteomIn general, relative quantitation strategies are used for discovery-based clinical proteomics. Once proteins of interest have been identified they require further validation. While antibody-based techniques such as ELISA fulfill this role, MS-based targeted assays are well suited for validation, especially if no suitable antibodies are available. The most commonly used method is called multiple-reaction monitoring mass spectrometry (MRM-MS). This assay is well established for small molecules and peptides and is carried out using triple quadrupole mass analyzers. MRM-MS assays require prior knowledge of the parent ion (MS1) and fragment ion (MS/MS) mass-to-charge ratios. The combination of a parent ion and 3–5 associated fragment ions, termed transitions, are selected by the quadrupole. Quantitative data is obtained by measuring the area under the curve of the transitions extracted ion chromatograms. More recently, targeted assays have also been developed using quadrupole Orbitrap mass analyzers—termed parallel reaction monitoring mass spectrometry (PRM-MS), which leverage the high-resolution, accurate-mass (HRAM) Orbitrap for increased specificity , 86. SimIn the context of clinical proteomics, tissue analysis provides the most accurate reflection of the tumour’s physiological state. As mentioned, recent advancements in LC–MS technology have continuously enabled increased proteome coverage with reliable quantitation. These studies now enable routine proteomics analyses of patient tumor tissues for biomarker discovery, discovery of biological pathways and integrations with available genomics/transcriptomics profiles.Tissue-based proteomic strategies have been applied to the study of many cancer types, including prostate , 91, breA major application of discovery-based proteomics in cancer tissues is the development of risk stratification and cancer subtyping systems. This has been demonstrated by label-free strategies with several recent examples of DIA applications. Bouchal et al.  performeAforementioned label-dependent protein quantitation techniques have also been applied to tissue proteomic for improved risk stratification. Iglesias-Gato et al.  performeAside from improving risk stratification, and thus patient survival, clinical proteomics can be used to identify new targets for improved treatment efficiency. Recently, a label-free, global proteomics approach was used to investigate the proteomes of 25 FFPE ovarian cancer tissues, stratified by chemo-resistance. More than 9000 proteins were detected and the differential expression of CT45 was related to a positive chemotherapy response. Little was known about CT45’s molecular function, thus phosphoproteomic analysis of cell line models was pursued and suggested that CT45 increases the repair response to DNA damage. Additionally, CT45 produces immunogenic peptides that recruit cytotoxic T cells and promote tumour killing. Therefore, clinical proteomics was a powerful tool in identifying a target for future immunotherapies .N-glycopeptides followed by reversed-phase LC fractionation prior to shotgun MS analysis. Different protein and glycoprotein signatures were found in ADC and SqCC samples, with pathways distinguishing between tumour types.As demonstrated in the study above, tissue global proteomic studies can be further extended to the investigation of PTMs. Yang et al.  studied Protein kinases play an important role in signal transduction pathways, and the dysregulation of these pathways is an intriguing area of study in cancer . Modern Tissue proteomic studies to date have focused on discovery-based studies that highlight countless potential markers of cancer. We need to move towards rigorous validation of these targets if the field is to evolve towards routine analysis in the oncology clinic. This bridge is being formed by targeted MS methods. PRM of tissue lysates, or immuno-affinity pulldowns of specific proteins from lysates, will become more mainstream . For exa2 correlation of 0.88. Phosphoproteomic results, with an average of 37,000 phosphosites quantified per sample, were less reproducible. The maximum deviation in phosphoproteome coverage was 24% with a R2 correlation of 0.72 [2 = 0.8) [When using the same sample preparation methods, LC–MS configuration and data analysis pipelines, considerable inter-lab reproducibility can be achieved. TMT-10 was used to multiplex PDX tissues derived from two breast cancer subtypes and compared across three independent laboratories. Overall, 7700 human-originating proteins were distinguishable from the 3100 mouse-derived stromal proteins with a maximum deviation across laboratories of 7%. Additionally, the TMT ratio between the two subtypes produced a minimum R of 0.72 . Similar of 0.72 . Another2 = 0.8) . Of cour2 = 0.8) . Cross-r2 = 0.8)  instillsOne factor to consider in quantifying changes in the tissue proteome is tumour heterogeneity. This was recently shown by sampling the proteome of frozen prostatectomy specimens by SWATH-MS from benign prostatic hyperplasia and adenocarcinoma patients. To account for inter-patient, intra-tissue and inter-tissue variability, tissue biopsy sections were investigated from different areas of the same sample, different samples and different patients. It was noticed that proteins involved in DNA repair pathways differ significantly between different areas of the same tumor tissue, owing to tumour heterogeneity. Thus, it was suggested to account for this variability in future studies when identifying protein biomarkers from tissues . EffortsAnother MS-based technique that provides spatial information and shows potential in the clinic is Mass Spectrometry Imaging (MSI). MSI has already been established in the detection of metabolomics-based markers, as is making strides in the proteomics sphere. A sectioned tissue biopsy is surveyed by MALDI-MS at ever-increasing spatial resolutions, as low as a few micrometers , and rou+ tumor-associated macrophages [This tumour heterogeneity creates the need for single-cell resolution in proteomics. Fluorescence-activated cell sorting (FACS) is an antibody-based technique used to sort a heterogeneous mixture of cells based on the expression of predetermined protein markers but comes with low multiplexability of markers. An MS-based alternative has emerged, called mass cytometry, and allows for dozens of protein markers to be monitored in individual cells. This is accomplished by coupling antibody probes to unique stable heavy metal isotopes rather than fluorophores . The celrophages . The masrophages  or more rophages .Potentially the most powerful application of tissue proteomics to cancer research is using it in a concerted effort to complement genomics. This rapidly growing field is termed proteogenomics , 131. GeWhile the cancer genome and transcriptome of many cancers have been well elucidated, the cancer proteome and its relation to up-stream genomic alterations are poorly documented. In recent years, a growing number of studies have begun to integrate all levels of omics data to describe a comprehensive multi-omic assessment of tumours. These projects often require large collaborations between laboratories with unique skill sets, such as The Clinical Proteomic Tumor Analysis Consortium (CPTAC). This network was created by the National Cancer Institute to promote collaboration in an effort to accelerate our understanding of the molecular basis of cancer, with an initial focus on ovarian, breast and CRC . The consortium has produced increasingly complex multi-omic datasets, using standardized methods and applying them to growing cohort sizes of diverse surgical tissues. Stemming from the consortium, the Applied Proteogenomics Organizational Learning and Outcomes (APOLLO) network was created to bridge the CPTAC findings so that proteogenomic analysis becomes a routine component of personalized medicine. The International Cancer Proteogenome Consortium (ICPC) also works to bring cancer researchers together to share and compare data across 10 countries. The benefit of these networks is the validation of results in multiple centres. Irreproducibility in proteomics arises from differences in peptide digestion, pre-fractionation, chromatography, MS configuration and bioinformatics . These aProteogenomic studies on breast cancer have recently demonstrated the ability to narrow down candidates for driver genes and to identify therapeutic targets. Mertins et al.  quantifiHuman hepatocellular carcinoma has been investigated by a couple of proteogenomic studies. In collaboration with CPTAC, Gao et al.  applied Sinha et al.  investigOther notable cancer proteogenomic studies include the study of medulloblastoma , early-oThe promise of liquid biopsies is that they are thought to provide proteomic information representative of a given tumour or tissue type but can be collected in a less-invasive and longitudinal manner. In clinical laboratory assays, blood is the most widely used human body fluid in disease diagnosis, prognosis and treatment outcomes. Blood consists of cellular components  and a liquid component called plasma . Blood iImmunodepletion and fractionation has expanded the number of detected plasma proteins into the thousands . ImmunodAfter establishing the human proteome atlas project (HUPO) in 2001 , the HumVarious studies have investigated the blood (plasma and serum) proteome from patients with various types of cancer, as summarized in Table In a squamous cell lung carcinoma study, blood samples coming from the pulmonary artery and vein of noncancerous and cancerous lung regions were obtained during surgery. The plasma was immunodepleted and fractionated and peptides were quantified by iTRAQ. The results showed 50 proteins to be abundant in the vein draining part of the cancerous regions compared to the noncancerous sectors . In anotCirculating tumor cells (CTC) are tumor cells that shed from the primary tumor into the circulatory system and can lead to metastasis in different organs, but provide a unique source of potential biomarkers . The occN-glycoproteome during PCa progression. The N-glycopeptides from serum and prostate tissue of wild-type and KO (PTEN) mice models were enriched and analysed by LC–MS. This generated a total of 757 N-Glycoproteins. The comparison between WT vs KO sera and tissue shortlisted 49 biomarker candidates. These candidate proteins were selected for SRM-based targeted assay in 143 patient serum samples (disease and control). In the end, 33 proteins were quantified by SRM in 80–105 patient samples. An additional nine proteins were validated by ELISA, totalling 39 proteins as potential PCa biomarkers.The plasma glycoproteome from various carcinomas were studied by Sajic et al. . In thisN-glycopeptides were enriched from serum and tumor tissues using hydrazide chemistry and analyzed by label-free proteomics. This resulted in 3675 N-glycopeptides corresponding to 2200 proteins containing the Asn-X-Ser/Thr N-glycosylation sequon. Following species-assignment and bioinformatic prioritization the authors systematically developed targeted proteomics assays and applied them to two longitudinal cohorts of HGSC serum samples. This study reports four putative biomarkers for the early detection of HGSC recurrence. The study reports on a novel strategy for the discovery of tumor-derived proteins using a combination of N-glycoproteomics and PDX models. A similar study was performed by Hüttenhain et al. [In a recent study by Sinha et al. , potentin et al. . GeneticUrine is another commonly sampled human body fluid because it is produced in large volumes and can be easily collected in a non-invasive manner. From a proteome perspective, urine is less complex than blood, with a narrower dynamic range, and is less prone to proteolytic degradation allowing for more stable storage over longer periods of time . ImproveIt is estimated that 70% of urine proteins originate from the kidney and urinary tracts. As such, this proximity makes urine a valuable resource for monitoring urinary tract cancers . CurrentThe remaining 30% of urinary proteins are from the glomerular filtration of blood suggesting that urine can also provide insight into cancers of distant organs . A multiSince the urethra passes through the prostate, urine is a valuable source of PCa biomarkers. A desirable trait of PCa markers is that they originate from the prostate rather than from other organs in the male urogenital tract. The Early Detection Research Network (EDRN) has studied post-DRE  urines which has been shown to contain a trove of potential biomarkers indicative of PCa status, including a non-coding RNA transcript, PCA3 . HoweverUrinary extracellular vesicles (EVs) have attracted significant interest in recent years . AlthougN-glycoproteome related to epithelial ovarian cancer [Aside from blood and urine, there are a variety of alternative human body fluids that could potentially be used for biomarker discovery. Some of these non-conventional fluid samples are rich sources of organ-specific proteins due to their close proximity, but often come at a cost of invasive sample collection. Thus, they may not be as applicable to routine clinical practices such as early cancer detection or longitudinal monitoring of cancer progression. The aforementioned prostatic secretions (often referred to as expressed prostatic secretion—EPS) is a fluid naturally produced by the prostate. The EPS proteome is hence a rich source of prostate-derived proteins and this fluid’s proteome has been directly profiled in the absence of a urinary background , 193–195n cancer , and then cancer .Meanwhile, there exists a subset of alternative bodily fluid types that can be collected more readily but their proteomes have not been characterized as thoroughly as more conventional body fluids. Tears provide an intriguing biomarker source and only recently have there been efforts to characterize its proteome . Tears fMoving forward, supplementary inter- and intra-patient variability studies will be needed to confirm proper sample collection protocols and patient selection. As clinical fluid sample preparation methods become more established and standardized, these alternative body fluid proteomes will be characterized in further detail as part of the search for robust non-invasive cancer biomarkers.In summary, recent advances in biobanking and proteomics technologies now enable the robust profiling of clinical samples to unprecedented depth. To become a mainstream technology in clinical laboratories, similar to well-established genomics assays, proteomics technologies and investigators must embark on large-scale, possibly multi-institutional validations studies. Targeted proteomics assays are likely to play a central role for the validation of tissue or fluid-based biomarkers. Technologies such as MRM-MS (or more recently PRM-MS) are already firmly established in clinical biochemistry laboratories around the world for the detection and quantification of small molecules. The establishment of protein assays is the next logical step, but requires rigorous assay development metrics and large richly annotated validation cohorts.Recent proteogenomics studies have also demonstrated the complementarity of proteomics and genomics technologies. Interrogation of biomolecules along the central dogma are expected to provide novel biological insights, multi-omics biomarkers and possible novel drug targets. While a handful of impressive studies have been published in recent years these have mainly focused on technical aspects of proteogenomics. What is missing is proteogenomics studies with a clear clinical question and large tissue cohorts to arrive at statistically powered conclusions. Another significant bottleneck of proteogenomics is the current lack of appropriate analyses strategies. While the field is capable of generating these large datasets, computational analysis strategies will require further improvements. This will require close collaboration between genomics, proteomics and data science/statistics investigators.A unique feature of proteomics technologies is the ability to detect subcellular localizations , proteinMoving forward, the field of clinical proteomics is likely to see a rapid expansion of clinical cohort sizes as a result of standardized, high-throughput sample preparation techniques. This will minimize the frequency of studies that suffer from statistical underpowering and improve efficiency of translating biomarker candidates and drug targets to clinical application. Proteomics will increasingly become a critical part of cancer systems biology that integrate multi-omics data from genomics, epigenomics, transcriptomics and PTMs. This will create demands for superior computing power to handle and analyze increasingly large amounts of data. Further improvements in MS instrument sensitivity and speed will make deep proteome coverage more regularly attainable, especially without the need for extensive pre-fractionation. Improvements in detection/quantitation levels will also allow clinical proteomics to expand towards minimal input material and single-cell proteomics. Finally, data analysis pipeline will continue to enable the detection of protein panels and signatures that provide more diagnostics and prognostic accuracy relative to singular markers. These advancements will all be required for MS-based clinical proteomics to reach its full potential in translating research discoveries to improvements to clinical practice.


Inflammatory bowel disease (IBD) is a group of chronic disorders that cause relapsing inflammation in the gastrointestinal tract and comprise three major subgroups of Crohn's disease (CD), ulcerative colitis (UC), and IBD-unclassified (IBDU). Recent advances in genomic technologies have furthered our understanding of IBD pathogenesis. It includes differentiation rare monogenic disorders exhibiting IBD and IBD-like inflammation (monogenic IBD) from patients with the common polygenic form of IBD. Several novel genes responsible for monogenic IBD have been elucidated, and the number of reports has increased due to advancements in molecular functional analysis. Identification of these pathogenic genetic mutations has helped in elucidating the details of the immune response associated with gastrointestinal inflammation and in providing individualized treatments for patients with severe IBD that is often unresponsive to conventional therapy. The majority of monogenic IBD studies have focused on young children diagnosed <6 years of age (very early-onset IBD); however, a recent study revealed high prevalence of monogenic IBD in older children aged >6 years of age as well. Meanwhile, although patients with monogenic IBD generally show co-morbidities and/or extraintestinal manifestation at the time of diagnosis, cases of IBD developing as the initial symptom with unremarkable prodromal symptoms have been reported. It is crucial that the physicians properly match genetic analytical data with clinical diagnosis and/or differential diagnosis. In this review, we summarize the essential clues that may physicians make a correct diagnosis of monogenic disease, including classification, prevalence and clinical phenotype based on available literatures. Inflammatory bowel disease (IBD) is a group of chronic disorders that cause relapsing inflammation in the gastrointestinal tract. They traditionally comprise three major subgroups: Crohn's disease (CD), ulcerative colitis (UC), and IBD-unclassified (IBDU) . Recent Recent advances in genomic technologies, such as next-generation sequencing, have made it possible to diagnose severe refractory IBD and IBD-like disease as rare monogenic disorders. IBD and IBD-like diseases caused by monogenic disorders transmitted according to Mendelian inheritance patterns have been described as “monogenic” IBD, in contrast to “polygenic,” or classical IBD. The majority of monogenic IBD cases occur in children diagnosed under 6 years of age. Glocker et al. first identified interleukin IL-10 receptor deficiencies in young infants with severe IBD and perianal disease . Since tThe recent advances in translational research into monogenic IBD has resulted in three major contributions to IBD pathogenesis and care including: (1) increased knowledge of pathogenesis of disease, (2) better phenotyping, (3) targeted therapy based on genetic variants . First, Recent developments in scientific technology have led to the production of new drugs affecting the immunological pathways of intractable monogenic IBD. Using a phenotypic high-throughput drug screen, Jardine et al. showed that leflunomide could be used in TTC7A-deficient patient-derived colonoids. Clinical trial are currently underway .The impact of the genetics for classical IBD (polygenic IBD) is also significant and different from that of monogenic IBD. Genome-wide association studies (GWAS) have identified about 240 disease loci linked to classical adult-onset IBD and have substantially expanded our understanding of the genetic architecture and causative mechanisms of IBD  19, 20), 2019, 2While these GWAS studies have identified important pathways associated with IBD including autophagy, a clear understanding of the interactions of IBD risk loci and causal genes as most of these GWAS loci are in introns or intergenic regions. Additionally, the majority of common genetic risk polymorphisms detected by GWAS have small effect (odds ratio <1.5). Statistical analysis indicated that these variants could explain only 13% and 8% of the variance in disease heritability for CD and UC, respectively . In the Variable expressivity is defined as the degree of variation of the clinical phenotype in individuals with monogenic disorders. Expressivity is different from penetrance, a term referring to whether the patient has the clinical phenotype associated with a genetic modification . The expIncomplete expressivity is assumed to result from the effects of unlinked modifier genes, epigenetic changes, or environmental factors . RecentlMonogenic IBD usually exhibits a Mendelian inheritance pattern, but some monogenic IBD disorders that are inherited as X-linked or autosomal recessive  may haveAutosomal recessive forms of inheritance are different from X-linked inheritance. When using whole exome sequences (WES), we rarely find only single-allele mutations causing monogenic disorders, although the phenotypes may be compatible with monogenic disorders. There are potential explanations for this phenomenon: (1) the existence of copy-number variation or low frequency gene mosaicism, which may be missed by WES, (2) a second-hit allele may be located in an intron or a non-coding region, altering splice site recognition; or (3) unknown modifier genes may affect penetrance. Regardless of the explanation, an approach to determine whether a variant is disease causing, such as additional sequencing and functional analysis are necessary . For exaEven if only a single-allele mutation causing monogenic disorder is found, the clinicians should consider additional functional test if the patient's phenotype is consistent with that disorder.In recent years, several cohort studies have been conducted, using WES and targeted genome panel sequence (TGPS) to determine the prevalence of monogenic IBD in children  35–41).–41.35–41Recently, Crowley et al. reported data from a cohort of over 1,000 children with IBD at a single center in Canada . This ceThe majority of the studies on monogenic IBD have focused on children younger than 6 years of age. Recently, there have been several reports dealing with children older than 6 years of age. The aforementioned large cohort from Canada showed that 2.5% of pediatric IBD occurring between the ages of 6 and 18 years was monogenic IBD . Three oThe key indicators of monogenic IBD in clinical practice are age of onset, family history, and extraintestinal manifestations based on individual gene defects. Infant IBD (under 2 years of age at onset) and VEOIBD are more likely to be monogenic IBD, and aggressive genetic testing is recommended. Concerning family history, there are two issues to be aware of. The first is “family relationships” including consanguinity. The second is “family diseases” as it is not uncommon for disease presentation to differ among family members despite having the same gene defect . TherefoIn the following sections, we discuss comorbidities. Extraintestinal manifestation is a critical indicator of monogenic IBD. Physical findings and comorbidities of which physicians should be aware at the initial physical examination and during follow-up are shown in We sometimes have difficulty diagnosing monogenic IBD because of the lack of severe complications. The most frequent disorders in this category are XIAP deficiency and CGD , 58, 59.Regarding XIAP deficiency, flow cytometry to evaluate XIAP expression is valuable for diagnostic screening tests. However, there are patients with XIAP deficiency who may have a normal protein expression but lack function. Therefore, we need assays for evaluating the TNF production of monocytes stimulated by L18-MDP (muramyl dipeptides) . For theEarly diagnosis is beneficial for patients with both XIAP deficiency and CGD. XIAP can develop life-threatening hemophagocytic lymphohistiocytosis (HLH), therefore HSCT should be considered early before HLH occurs , 61. CGDhttp://www.neopics.org/) and (http://www.VEOIBD.org) keep working to identify the causes and to develop new treatments together with international pediatric gastroenterologists and scientists from academic centers around the world.In this paper, we have reviewed the essential features of monogenic IBD, as seen in clinical practice. The use of WES and TGPS has led to improved diagnosis of severe refractory IBD as monogenic IBD. In the future, more genetic mutations will be discovered, and the role of immunity in intestinal inflammation will be elucidated using techniques including transcriptomics, proteomics, and metabolomics. It is further expected that appropriate treatments for these monogenic disorders will be developed. Because monogenic IBD is very rare, the collaboration of specialized facilities around the world is required for the further development of individualized treatment strategies. The interNational Early Onset Pediatric IBD Cohort Study (NEOPICS) (RN conceived, designed, and drafted manuscript. AM drafted, edited, and revised manuscript. RN and AM approved final version of manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The onset of chemotherapy-induced peripheral neurotoxicity (CIPN) is a leading cause of the dose reduction or discontinuation of cancer treatment due to sensory symptoms. Paclitaxel (PTX) can cause painful peripheral neuropathy, with a negative impact on cancer survivors’ quality of life. While recent studies have shown that neuroinflammation is involved in PTX-induced peripheral neurotoxicity (PIPN), the pathophysiology of this disabling side effect remains largely unclear and no effective therapies are available. Therefore, here we investigated the effects of human intravenous immunoglobulin (IVIg) on a PIPN rat model. PTX-treated rats showed mechanical allodynia and neurophysiological alterations consistent with a severe sensory axonal polyneuropathy. In addition, morphological evaluation showed a reduction of intra-epidermal nerve fiber (IENF) density and evidenced axonopathy with macrophage infiltration, which was more prominent in the distal segment of caudal nerves. Three weeks after the last PTX injection, mechanical allodynia was still present in PTX-treated rats, while the full recovery in the group of animals co-treated with IVIg was observed. At the pathological level, this behavioral result was paralleled by prevention of the reduction in IENF density induced by PTX in IVIg co-treated rats. These results suggest that the immunomodulating effect of IVIg co-treatment can alleviate PIPN neurotoxic manifestations, probably through a partial reduction of neuroinflammation. Paclitaxel (PTX) is a very effective anti-tubulin drug belonging to the family of taxanes. It is largely employed in the treatment of many solid tumors including breast, prostate, non-small cell lung, pancreatic and gynecological cancers . DespiteDespite remarkable efforts at the preclinical level, a comprehensive knowledge of the mechanisms leading to PIPN is still lacking. PTX dependent inhibition of tubulin depolymerization leading to microtubule dysfunction seems to be the most reasonable hypothesis , althougIn fact, several studies reported an increase of pro-inflammatory cytokines and chemokines in the plasma, serum, dorsal root ganglia (DRG) neurons, sciatic nerves, skin of the hind paw and spinal cord of PTX-treated rodents . These aHuman intravenous immunoglobulin (IVIg) are therapeutic polyspecific IgGs derived from plasma pools of thousands of healthy donors, characterized by multiple immunomodulatory and anti-inflammatory properties. IVIg are used to manage neuropathic pain from various neurological disorders . While tPTX and PTX-IVIg co-treatment were well tolerated by the animals during the experiment, it did not induce any significant difference in body weight compared to vehicle-treated rats and no animals died during the study nor showed signs of distress.Serum concentrations of IVIg were measured in each IVIg-treated group over the entire sampling period, i.e., until follow up in all animals a. At eacp < 0.001; p < 0.01; At baseline, no significant difference in the mean withdrawal threshold was observed among the groups. At the end of the treatment, PTX, PTX + IVIg2, PTX + IVIg4 groups showed a significant decrease in the mechanical threshold compared to VEH-treated rats , whereas no significant effects were observed in NCV. At the end of follow up, a significant decrease in proximal caudal SNAP amplitude and NCV was observed in PTX-treated rats. The analysis performed in PTX and IVIg co-treated animals did not show any significant protective effects of IVIg administration on neurophysiological parameters  showed attenuated macrophage infiltration if compared to the PTX group .No macrophage infiltration was observed in sciatic nerve and DRG samples of PTX and PTX + IVIg co-treated rats compared to VEH.PIPN is a common and potentially severe adverse event in patients undergoing PTX chemotherapy regimen  that mayIn the last decade, activation of immune and immune-like cells in both the peripheral and central nervous systems have gained rising interest as one of the putative mechanisms responsible for PIPN onset and progression. The results of clinical trials indicate that IVIg administration is effective in reducing pain in chronic conditions where neuropathic pain is prominent . FurtherFurthermore, IVIg could modulate macrophage functions, interfering with the production of the macrophage pro-inflammatory factor involved in peripheral nerve degeneration and exerting their immunomodulatory effects acting on several components of the immune system, as observed in chronic inflammatory demyelinating polyneuropathy . HoweverDespite a difference at the pathological level, the protective effect of IVIg was not accompanied by an improvement in distal caudal NCV and SNAP, as already observed in the bortezomib-induced peripheral neurotoxicity model . A possiIn contrast to the protective effects observed, once the animals were co-treated during the entire PTX treatment, the morphological analysis did not show any evident protective effect of IVIg administration, starting at mid treatment of PTX regimen. This lack of effectiveness is not unexpected, since PIPN damage is detectable as early as after two weeks of treatment, as confirmed in experimental PIPN also by the demonstration of a very early increase in neurofilament light chain, a specific biomarker of nerve damage  and infln = 2/3 animals per cage) with rodent diet and water ad libitum, subjected to daily monitoring of their clinical conditions and weighted twice a week for a general health check and drug dose adjustment. At each time point of sacrifice, rats were euthanized by CO2 inhalation followed by cervical dislocation. All experimental procedures were carried out in accordance with National Institute of Health guidelines for animal care and use of Laboratory animals  and approved by the Milano Bicocca University Ethics Committee (protocol n 7579/19).Female Wistar rats (175–200 g) were purchased from Envigo Laboratory . The animals were housed in an animal facility characterized by a standard light cycle of 12 h on and 12 h off, a constant temperature of 22 °C ± 2, and 50 % ± 20 relative humidity. Rats were housed in an adequate cage  dissolved in a vehicle solution composed of 10% tween 80, 10% EtOH absolute and 80% saline solution, as previously described in detail .n = 12), and three groups were treated with PTX alone  or in co-treatment with two different IVIg schedules  followed by follow up. IVIg were infused at the dosage of 1g/kg the day before PTX treatment at the specific time points described in the flow chart of the study , 48 female Wistar rats were randomized into 4 experimental groups: one group was treated with vehicle for 4 weeks followed by follow up , as previously described in detail . These dBriefly, rats were allocated in plexiglass cages placed on a metal grid floor for 15 min. After the acclimation period, a metal filament, exercising a linear increasing force ramp which reaches 50 g in 20 s, was applied to the plantar surface of the hind paw. Mechanical threshold force, i.e., the minimum pressure required to elicit a withdrawal reflex of the paw, was registered three times for each paw and it was automatically recorded by the instrument and then calculated as the average of six consecutive values (expressed in grams). This test was conducted in a controlled behavioral test room by a researcher who was blind to treatments. An upper limit cutoff of 20 s was fixed.After animal sacrifice sciatic nerves, caudal nerves and DRG samples were collected a and proTo evaluate a small-fiber peripheral nerve damage, intra-epidermal nerve fiber (IENF) density in the hind paw footpad was evaluated, as already reported . SamplesTo investigate the role of inflammation within DRGs, sciatic and caudal nerves, we determined the CD68 profile, a phagocytically active macrophage detector, on the samples collected after sacrifice as previously described . Brieflyp value < 0.05 was considered statistically significant. All statistical analyses were performed using the GraphPad Prism4 software .Statistical analyses of body weight, neurophysiological studies, dynamic test and IENF density were evaluated using the Kruskall-Wallis test followed by Dunn’s post hoc test for each time point. A The results of our study extend previous observations in the experimental bortezomib-induced peripheral neurotoxicity model  and indi