It is not clear whether the frequently reported phenomenon of exercise-induced anorexia is exacerbated or blunted in warm or cold environments. Therefore, this study investigated the effects of exercise in three different environmental temperatures vs. rest, on perceptions of appetite, appetite regulating hormones, and food intake.In a randomized repeated-measures design, 18 Canadian Armed Forces members  completed four 8-h trials in a thermally-controlled chamber: one 8-h resting trial at 21 °C (Sedentary); and three trials where participants completed two 2-h circuits of standardized military tasks interspersed with two 2-h rest periods, once at 30 °C (Hot), once at 21 °C (Temperate), and once at − 10 °C (Cold). Participants consumed military field rations ad libitum and had their appetite assessed with visual analogue scales. Plasma concentrations of GLP-1, PYY, acylated ghrelin, and leptin were also determined.p < 0.05). While neither exercise nor environmental temperature altered circulating GLP-1 levels, exercise in all environments increased blood concentrations of PYY (p < 0.05). Leptin concentrations were elevated in the heat and diminished in the cold (p < 0.05), and acylated ghrelin concentrations were affected by both exercise and ambient temperature resulting in Sedentary = Cold>Temperate = Hot (p < 0.05). Contrary to the changes in appetite perceptions and hormonal concentrations, dietary intake was not different between conditions (p > 0.05). Relative energy intake  on the other hand, was significantly higher during the Sedentary condition than it was during any of the active conditions (p < 0.05). Most (83%) of the participants were in a positive energy balance during the Sedentary condition, whereas during most (80%) of the active conditions  participants were in a negative energy balance.Appetite was perceived as being suppressed in the heat compared to the cold (In this study where food was freely available, variations in ambient temperature, exercise vs. rest, appetite-regulating hormone concentrations, and subjective appetite sensation were not associated with any changes in dietary intake within 24-h of acute, prolonged exercise. Several studies have assessed the acute effects of exercise on appetite, appetite-regulating hormone concentrations, and energy intake (EI) , 67, 69.It is thought that environmental temperature might also affect appetite, as the results of early cross-sectional military research suggests that food intake is low in the heat and high in cold environments . In humaAn understanding of how exercise in varying temperatures affects appetite and EI can be important for those who participate in outdoor sports, in occupational, and military operational settings requiring prolonged outdoor physical exertion. In military personnel for example, body weight loss due to under-eating during field training and deployed operations is not uncommon even when food is plentiful , 39, 61.Therefore, the purpose of this study was to investigate the effects of arduous physical activity in varying ambient temperatures on dietary intake, appetite sensation, and appetite-regulating hormone concentrations in military personnel throughout an eight hour day.n = 21) and female (n = 6) Canadian Armed Forces (CAF) members volunteered to participate in the study (Table n = 3), reconsideration (n = 3), or loss of contact (n = 1). Two participants were also excluded due to their non-compliance with the study restrictions. As a result, 18 participants  completed the study. All participants were free from metabolic and cardiac disorders and were not taking any medications or nutritional supplements. All participants were fully informed of the details, discomforts and risks associated with the experimental protocol before being asked for their written informed consent. Informed consent was obtained from all individual participants included in the study. The study protocol was reviewed and approved by institutional human research ethics committees at Defence Research and Development Canada (#2013–075), and the University of Toronto (#29914).Twenty-seven healthy, male n = 2 and fema. Participants made three initial visits to the lab, followed by an additional four for the experimental conditions.The study design is graphically depicted in Fig. 1V̇O2peak) measured using indirect calorimetry during a progressive incremental exercise test to voluntary exhaustion on a treadmill [V̇O2peak. During Visit 3, participants reported to the laboratory after a 10 h overnight fast, after having completed a three-day weighed food record to assess each participant’s typical dietary intake. Body mass was measured with a standard scale , and percent body fat was estimated via air-displacement plethysmography . Measurements were completed two consecutive times to confirm accuracy.Visit 1 consisted of the completion of the informed consent form, PAR-Q+ to clarify if health risks suggested precluding their engagement in arduous exercise , and thereadmill . Oxygen https://www.random.org/) determined which permutation a participant would be assigned to.Participants completed a total of four different experimental conditions; each condition started in the morning and lasted 8 h in the environmental chamber. At least one week intervened between conditions Fig. . For theAt 6:00 am on the morning of each experimental condition, participants swallowed a telemetric temperature capsule for monitoring of internal body temperature with 250 mL of water at home. At 7:45 am, participants reported to the laboratory for initial measurements of body weight measured using a standard scale , resting blood pressure using an automated blood pressure cuff , and core temperature using the Equivital™ . The visual analogue scales for appetite (VASA) (described in the measurements section) were also administered while fasting. Participants consumed breakfast, and then donned the temperature appropriate military clothing for the environmental condition on that day. Participants wore standardized military clothing items that are mandatory during field operations; these included: military fatigues, a tactical vest (~ 3-7 kg), a fragmentation protection vest (2-3 kg), combat boots (1.5 kg- 2.5 kg), and a helmet (~ 1.5 kg). Other than these required clothing, participants decided which military clothing to wear or not wear in order to be comfortable in the condition that they would be enduring . Participants also wore a portable metabolic system: the Metamax 3B , which weighed about 1.5 kg. This device required the participant to wear an oral-nasal mask over the face and an interface box strapped to the torso. A heart rate strap , that was compatible with the Metamax 3B unit, was also affixed to the participant’s chest, as was a device for the monitoring of internal body temperature from a telemetered ingestible temperature capsule . The participants then entered the environmental chamber in which room temperature and relative humidity were controlled for the day’s duration. The environmental chamber was 5.9 m long and 4.5 m wide.very light activity (< 50% max HR) 7% of the time, light activity (50 to < 64% max HR) 29% of the time, moderate activity (64 to < 77% max HR) 31% of the time, vigorous activity (77 to < 94% max HR) 26% of the time, and very vigorous (> 94% max HR) 6% of the time. After 8 h, participants exited the environmental chamber and took their supper ration – and any leftover items from their breakfast and lunch rations – home with them. Participants continued to document all food that was eaten upon leaving the laboratory and returned all waste and uneaten food the following day.Following entry into the chamber, participants either rested (Sedentary) or they completed the standardized military tasks . The infantry activities completed during the circuits included resting activities , and both aerobic , inclines , and while carrying different loads ) and strength / muscular endurance (moving sandbags (20 kg each), varying ammo cans (3.2 kg and 13.7 kg) and jerry cans , dragging an 85 kg dummy across the floor, building a sandbag barrier, etc.) activities. When classifying the intensity of the activities based on the percentage of maximal heart rate  was assWhere EE = energy expenditure, H=Hot condition, C=Cold condition, T = Temperate condition, S=Sedentary condition, BM = body mass in kg, and where 1 MET is equivalent to 1 kcal/kg/h [cal/kg/h .There were 18 different military field rations choices available to the participants , and the participants selected 1 breakfast, 1 lunch, and 1 dinner ration which was kept the same for all of the chamber days  for that participant. The three rations  provided 4093 ± 263 kcal and approximately 627 g of carbohydrate, 191 g of fat, and 108 g of protein. Each ration contained an entrée, dessert, sport drinks, bread, jam/peanut butter/honey, two hot beverages, condiments and depending on the ration, additional products such as chocolate bars and candy. In addition to the three rations, participants could take one snack ration pack  containing energy bars, sports drinks and other snack foods. Although military rations are provided as separate meals, items from any of these packs could be (and often are) consumed at any time. To determine the timing of food intake, the 8-h in the environmental chamber was segmented into 30 min intervals  to determine when participants chose to eat. Investigators documented all feeding behaviours  and weighed all food that was discarded. The CAF Directorate of Food Services provided (from the manufacturer) the nutritional information for all of the presented rations, some of which were based on chemical analyses. Dietary intake (EI and macronutrient intake) was determined by subtracting the energy and macronutrient content of the unconsumed food from the known quantities in the rations provided. Relative energy intake (REI) was calculated by subtracting estimated 24 h EE from the energy consumed  throughout the day. From REI, it was determined whether a participant was in a positive or negative 24-h energy balance.hunger, satiety, fullness and prospective eating. Subjective appetite was assessed while fasting, immediately following breakfast, and every hour throughout the 8 h in the environmental chamber . Blood samples were taken immediately upon arrival to the laboratory (time point 0), at the end of the first exercise bout (beginning of the first rest period) (time point 2 h), 30, 60, and 90 min into the first rest period , 120 min into the first rest period (immediately before the second exercise bout) (time point 4 h), at the end of the second exercise bout (beginning of the second rest period) (time point 6 h), 60 and 120 min into the second rest period (time point 7 h and 8 h respectively) while in the chamber  and 20uL of DPP-IV inhibitor . Samples for the determination of acylated ghrelin were collected in a chilled 2 mL K2EDTA blood collection tube that was injected with an 80uL 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) solution containing 2 mg of AEBSF [Four (4) mL were taken at each blood sample time point and assayed for the plasma concentrations of leptin, acylated ghrelin, GLP-1, and PYY. Samples for the determination of GLP-1, PYY, and leptin were collected in a chilled 2 mL Kof AEBSF . Once drof AEBSF .The concentrations of leptin, GLP-1, PYY, and acylated ghrelin in the blood samples were determined using electrochemiluminescent immunoassays (ECLIA). Three different Meso Scale Diagnostics (MSD) assays were run, one for the determination of GLP-1 and leptin , one for the determination of PYY , and one for the determination of acylated ghrelin . The sensitivity of the assays was 0.3 pg/mL for GLP-1, 56 pg/mL for leptin,13 pg/mL for PYY and 9 pg/mL for acylated ghrelin. The inter- and intra-assay coefficients of variation were 12 and 9% for GLP-1, 13 and < 5% for leptin, 8 and 6% for PYY and 14 and 9% for acylated ghrelin respectively. All samples for one participant were run in duplicate on the same assay; as a result the effects of inter-assay variation were controlled within participants.p < 0.05.Two-factor, repeated measures ANOVAs were used to examine differences between conditions over time for appetite sensation, EE, dietary intake, and REI. Mauchly’s Test was used to test the assumption of sphericity, and where Mauchly’s test was significant, the Greenhouse-Geisser estimate epsilon was assessed. If epsilon< 0.75, then the degrees of freedom were corrected using the Greenhouse-Geisser correction; if epsilon> 0.75, then the degrees of freedom were corrected using the Huynh-Feldt correction . Where sp > 0.05)[p < 0.05.Dietary intake (both EI and macronutrient intake) for the two days prior to each chamber day was not significantly different between conditions , nor wasp > 0.05). There wAs expected, the measured EE was significantly lower during the Sedentary condition compared to the others, and approximately 4-times greater during the 4 h of standardized military tasks compared to the same 4 h during the Sedentary condition (p < 0.05). The estimated daily EE was 56–58% greater (p < 0.05) during the different active conditions compared to the Sedentary condition Table . There wp < 0.05) . REI wap < 0.05). More energy was consumed at breakfast  and dinner  than at any of the sixteen 30-min intervals in the chamber (p < 0.05)   Fig. . During 05) Fig. . The amoig. Fig. . The indst 856 ± 93 kcal ahunger, fullness, satisfaction, and prospective eating (p < 0.05). Fullness and satisfaction scores shared a similar trend over time and participants felt the least full and the least satisfied upon arrival to the lab (following a > 10 h overnight fast), and they felt the most full and the most satisfied following breakfast. Hunger and prospective eating scores on the other hand, while being similar to each other, were contrary to fullness and satisfaction scores. The highest hunger and prospective eating scores occurred upon arrival to the lab, and the lowest hunger and prospective eating scores were found following breakfast . Participants were significantly hungrier during the Cold condition than they were during the Hot condition (Cold = 4.29 ± 1.35 vs. Hot = 3.31 ± 1.35) (p < 0.05). They felt significantly fuller and more satisfied during the Hot condition than they did during the Cold condition  (p < 0.05). There was also a significant difference in how much participants felt they could eat (prospective eating); they felt that they could eat more during both the Sedentary and Cold conditions, as compared to the Hot condition . There were no other significant differences between the conditions. There were also no significant interactions between time and condition for any of the appetite indices. When assessing the AUC0-8h for hunger, fullness, satisfaction, and prospective eating sensation the same trends emerged , and the amount of energy  and macronutrients  consumed in the 30 min following the assessment of subjective appetite. Medium positive correlations were found between hunger and the amount of energy  and nutrients (protein (g): r = 0.40, n = 645; carbohydrate (g): r = 0.39, n = 645; fat (g): r = 0.41, n = 645) consumed 30 min following appetite sensation assessment (p < 0.05). Similar correlations were also found between prospective eating and food consumed ; protein (g): r = 0.41, n = 645; carbohydrate (g): r = 0.40, n = 645; fat (g): r = 0.40, n = 645) (p < 0.05). On the other hand, medium negative correlations were found between fullness and all measures regarding energy  and nutrient intake (protein (g): r = − 0.43, n = 645; carbohydrate (g): r = − 0.43, n = 645; fat (g): r = − 0.45, n = 645) (p < 0.05), as well as between satisfaction and the amount of energy  and nutrients consumed (protein (g): r = − 0.45, n = 645; carbohydrate (g): r = − 0.46, n = 645; fat (g): r = − 0.47, n = 645) (p < 0.05).Pearson product-moment correlations were calculated among the four indices of appetite sensation .GLP-1 concentrations changed quite considerably over time and several significant differences were observed Fig. a. Only tn = 4), leptin concentrations were not different over time (data not shown).No significant differences were measured when assessing leptin levels over time when all  participants were included (data not shown). However, leptin concentrations are known to be different between males and females even after accounting for fat mass differences between the sexes . As a rep < 0.05). Concentrations of PYY were higher at 4 h post chamber entrance, than they were at 0, 2.5, 3, 3.5, 6, 7, and 8 h following chamber entrance (p < 0.05). Figure PYY levels upon arrival to the lab were significantly lower than all time-points Fig. c  Fig. d. ImmediRegardless of whether the results were analyzed using the concentration values attained, or as a percent change from average fasting values (data not shown), the same trends emerged.No significant differences between conditions were observed for GLP-1 Fig. a.Fig. 8ANo significant differences between conditions were measured for leptin when data for men and women was analyzed together (data not shown). When analyzed separately, leptin concentrations in males were significantly higher during the Hot condition (2494.1 ± 351.6 pg/mL) as compared to all other conditions: Cold (1825.8 ± 352.0 pg/mL), Temperate (2016.0 ± 352.2 pg/mL), or Sedentary (2192.4 ± 351.6 pg/mL) (p < 0.05). In males, leptin concentrations were also significantly higher during the Sedentary condition vs. the Cold condition  as compared to all other conditions: Cold (51.3 ± 2.3 pg/mL), Temperate (50.6 ± 2.3 pg/mL), or Hot (49.8 ± 2.3 pg/mL) (p < 0.05). No other significant differences were found .Acylated ghrelin levels were higher during both the Sedentary (113.1 ± 20.2 pg/mL) and Cold (102.2 ± 20.2 pg/mL) conditions than they were during either the Hot (83.4 ± 20.2 pg/mL) or Temperate (86.3 ± 20.2 pg/mL) conditions (p < 0.05). No significant differences were observed between Sedentary and Cold conditions, or between Hot and Temperate conditions . No significant differences were found between conditions for the AUC0-8h for any other appetite hormone.Acylated ghrelin concentrations were significantly lower during the Temperate  condition than during either the Cold  or Sedentary  conditions  but not body mass. No other hormones were correlated with any of the measured body stature or composition variables. When looking at correlations between the hormones of interest, there was a small but significant positive correlation between PYY and GLP-1 . Similarly a small positive correlation between acylated ghrelin and leptin  was found.As expected, fasting leptin levels were significantly higher in females (p < 0.05) and were significantly correlated to body fat percentage  and nutrients  consumed in 30 min following the blood sample collection. Table Similarly, when assessing the correlations between the appetite hormones and the different aspects of appetite sensation, weak correlations were detected between the hormones of interest and the four indices of appetite perception Table .Table 6PThe primary objective of this study was to determine the impact of arduous physical activity in varying ambient temperatures on appetite regulating hormone concentrations, subjective appetite sensations, and dietary intake. Although hormonal responses pointed towards appetite suppression with exercise , and subjective appetite sensation results suggested that appetite was highest in the cold, and lowest in the heat, actual dietary intake was the same regardless of whether the condition was sedentary or active and also regardless of the variations in ambient temperature.Interestingly, a substantial amount (4 h) of arduous exercise , even when completed in harsh ambient temperatures  did not alter dietary intake (both EI and macronutrient intake).Although previous studies have also reported no effect of acute exercise on subsequent EI, , 69, theAmbient temperature alone is thought to impact EI leading to increased food consumption as ambient temperature decreases and vice versa , 41. TheIn terms of energy balance, our study supports several other reports that acute exercise results in an acute negative energy balance , 67–69 iNotwithstanding these limitations, most of our participants (15 of 18) were in a positive energy balance during the Sedentary condition and a negative energy balance (11 of 18) throughout all three active conditions .It is important to note, that according to our food satisfaction surveys , the milIn contrast with previous studies , 44, 69,prospective eating, the suppression was evident when compared to the Sedentary condition as well. This is in agreement with Kojima et al. [hunger and motivation to eat were suppressed to a greater extent at 36 °C and 24 °C than they were at 12 °C, while Wasse et al. [hunger and prospective eating scores at 30 °C as compared to 20 °C, and lower satisfaction and fullness scores at 10 °C as compared to 20 °C. Consistent with these studies, the current study found a similar trend towards subjective appetite increasing in the cold, and diminishing in the heat.When evaluating the impact of ambient temperature, exercise in the heat suppressed subjective appetite more than during the Cold condition; for a et al.  who repoe et al.  found suNeither exercise alone, or in combination with a temperature challenge had any effect on circulating GLP-1 levels. Alternatively, exercise and ambient temperature induced hormonal changes in the direction expected to suppress appetite for both acylated ghrelin and PYY; exercise in both temperate and hot environments suppressed acylated ghrelin concentrations, while exercise in all temperature conditions increased circulating PYY levels.Although large changes in energy balance due to heavy exercise (e.g. marathon running) has previously been found to decrease leptin concentrations , leptin fullness and satisfaction, and positively correlated with prospective eating. While most previous studies have shown no relationship between blood leptin levels and subjective appetite sensations [fullness and satiety and negative correlations between leptin and both hunger and desire to eat while Vatansever-Ozen et al. [Interestingly, leptin was negatively correlated with nsations , 43, posnsations . Whethernsations  found pon et al.  found nofullness and satisfaction and negatively correlated with hunger and prospective eating. Although GLP-1 is not always found to be correlated with subjective appetite [Regarding GLP-1, most studies suggest that circulating GLP-1 levels increase following exercise , 66, 67,appetite , the resappetite , 65.Fig.fullness and satisfaction and negatively correlated with hunger and prospective eating. While PYY is considered to be a satiety hormone, most previous research has not found PYY to be correlated with any aspect of appetite sensation [In the present study, circulating PYY concentrations were increased similarly in the Temperate, Hot, and Cold conditions. Exercise typically increases circulating PYY levels , 66, 67,ensation , 33, 45.fullness and satisfaction, yet was not correlated with either hunger or prospective eating. While many appetite hormones are thought to impact appetite sensation, the correlations between these variables are not often observed [The first exercise bout decreased circulating acylated ghrelin concentrations in the present study, a finding which is consistent with earlier reports , 69, 71.observed , 25, 33.Overall exercise altered the hormonal milieu in a manner expected to suppress appetite, and ambient temperature only had minor additional effects beyond those associated with exercise alone.Although food accessibility during the measurement periods likely affected the timing of meals , variations in: ambient temperature, exercise, hormone concentrations, and subjective appetite sensation had no effect on the amount and timing of food intake, suggesting that none of these factors systematically change eating patterns in any predictable way. Dietary intake in our population was surprisingly similar across conditions. Interestingly, participants were generally unaware of how much they ate; when asked whether they thought they consumed more or less than usual, participants were only correct 35% of the time, 46% of the time they thought they ate less than they did, and 19% of the time they thought they ate more than they did. Finally, while menstrual cycle phase was documented in the present study, it was not controlled for, and the low number of women in the study prevented any further investigation into how the menstrual cycle may have impacted the results.It is likely that there are other factors not assessed in the current investigation that impacted appetite, such as “expected satiety”  (the lea


To evaluate mid‐term radiographic and functional outcomes of tibial plateau fracture (TPF) patients treated with the open reduction internal fixation (ORIF) technique and to find predictive factors of clinical outcomes.P < 0.05.A retrospective, single‐center study was performed enrolling a consecutive series of patients with diagnosis of TPF. All subjects were treated by ORIF between January 2010 and December 2015 at our level‐1 healthcare trauma center. The inclusion criteria were: age between 18 and 75 years; ORIF technique used for articular TPF, type 41‐B and 41‐C, isolated or with associated injuries. The patients were divided in two groups, according to fracture patterns and compared. Their characteristics, radiographic and clinical outcomes were recorded. Radiographs 12 months after surgery were evaluated for reduction and alignment, and radiographs at 24 months to describe post‐traumatic osteoarthritis (PTOA). Functional outcomes were assessed using the visual analog scale (VAS), the Short Form 36 (SF‐36), the knee injury and osteoarthritis outcome score (KOOS), and the American Knee Society score (AKSS) questionnaires. Return to work and sport activities were also investigated. Univariate and multivariate analysis were performed, and the statistical significance was defined as two‐tailed Forty‐five patients were included, 29 males and 16 females; the mean age was 54.5 years. The mean follow‐up was 57.18 months . There were AO 41‐B fractures  in more than half of the patients (66.67%), while the remaining 15 had AO 41‐C fractures (complete articular fractures). The sub‐type AO 41‐B3 was the most common, reported in 62.22% of patients. The mean KOOS score was 69.0. Mean AKSS and SF‐36 PCS scores were 79.0 and 41.4, respectively. There were significant relationships between age and functional results  and between BMI and KOOS Pain, ADL, Sport, and QoL. No differences were found between the two types of fractures regarding quality of reduction and alignment. AO 41‐C TPF tend to develop PTOA more frequently with respect to 41‐B fractures, while type AO 41‐C TPF had the worst clinical outcomes. We found that the presence of an articular step‐off and the malalignment of the tibial axis after surgery were predictive of daily pain felt by patients. PTOA was predictive of a worse AKSS. The overall complication rate was 13.33%: 1 superficial wound infection, 1 deep vein thrombosis, and 4 cases of transitory deficit of the common peroneal nerve.The present study demonstrates that early radiographic features may be predictive for pain perceived by patients at mid‐term follow‐up. Treatment strategies for complex TPF may include the combination of external and internal fixation. For example, the use of a wire external fixator allows fracture stabilization with minimal direct approach and a small surgical incisionDespite anatomical joint reconstruction, TPF are considered a major risk factor for post‐traumatic osteoarthritis (PTOA)et al. reported that 10 years after TPF surgery, 7.3% of the patients had a total knee arthroplasty. This corresponds to a 5.3 time increase in likelihood compared to a matched group from the general populationInterestingly, Wasserstein As most of the patients with TPF are active and highly demanding, an important issue is the incidence of OA, which can markedly impact the quality of life and affect the return both to sports and work activities. For these reasons, patients should be informed of the risk of developing PTOA, which could require future surgical intervention.Hence, the primary aim of this retrospective study was to evaluate the mid‐term radiographic and functional outcomes of patients surgically treated by ORIF using locking plates for partial articular (AO 41 type B) and complete articular (AO 41 type C) TPF. The second aim was to investigate whether radiographic features 12 months after surgery and the development of PTOA at 24 months were predictive of clinical‐functional outcome at last follow‐up.Between January 2010 and December 2015, 63 consecutive patients with the same number of TPF underwent ORIF surgical procedures at our level‐1 healthcare trauma center, a 1572‐bed multi‐disciplinary and multi‐specialty regional university teaching hospital. All subjects participating in this retrospective and experimental study received a thorough explanation of the risks and benefits of inclusion and gave their oral and written informed consent to publish the data. The study was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki as revised in 2000. The Local Ethical Committee approved the study . The patients received a thorough explanation of this study and gave their oral and written informed consent to be included in this analysis.The inclusion criteria were as follows: (i) patients' aged between 18 and 75 years old at the time of the trauma, admitted to our trauma unit in the selected period; (ii) ORIF technique used for articular TPF, isolated or with associated fractures, type 41‐B and 41‐C, according to Muller‐AO classificationFor this study, specific patient exclusion criteria were as follows: (i) pediatric patients; (ii) history of previous foot surgery or trauma; (iii) diagnosis of diabetes mellitus; (iv) cancer patients receiving chemotherapy and radiotherapy or with bone metastases; (v) patients with severe orthopaedic comorbidities ; and (vi) patients with untraceable postoperative radiographs.All patients were operated on by an orthopaedic surgeon of our trauma team (the senior authors) with the help of two residents. A tourniquet was not routinely used during synthesis procedures.Anesthesia and position: all anesthesiological procedures were performed with an ultrasound‐guided peripheral nerve block with atropine injections in combination with analgosedation. For isolated lateral TPF, a peripheral nerve bi‐block  was used, while in medial or bi‐condylar TPF, an ultrasound‐guided adductor canal block or laryngeal mask airway with deep sedation were also performed. Patients were in supine position on the operating table with the injured knee flexed approximately 60°. Prophylactic cefazolin (2 g) was administered and continued 24 h after surgery, while postoperative antithrombotic therapy (natriumenoxaparin) was started the same evening after the operation until weight‐bearing. During surgery, an image intensifier was used to enable optimal anatomical reduction and alignment. Temporary external fixation was used only when soft tissue conditions precluded definitive internal fixation.Approach and exposure: for unicondylar lateral TPF, a straight or hockey stick anterolateral incision (parapatellar) was used for fracture exposure. Medial column restoration was performed first with either a medial incision starting on the posteromedial border of the tibial metaphysis or a more central incision centered on the tibial tuberosity. For bicondylar fractures, a double incision was made: one posteromedial  and one anterolateral (peripatellar incision). In both groups, the following standardized surgical steps were taken.Reduction and stabilization: the knee joint was opened by sub‐meniscal arthrotomy to visualize fracture characteristics and any damage to ligaments and meniscus; reduction of the fragment and temporary fixation was done using K‐wire; depressed fragments were elevated and supported with a compression clamp or temporary K‐wires to obtain anatomical reduction, while bony defects were filled with synthetic tricalcium phosphate or with autologous bone grafts from the iliac crest.Internal fixation: depending on the fracture patterns, single or dual locking plates (LCP DePuy Synthes) were used to achieve definitive osteosynthesis.Postoperative protocol: all patients followed the same postoperative protocol and were checked in the same standardized manner by the same trauma team. Postoperative passive and active knee motion was encouraged the day after surgery to stimulate ROM. Toe‐touch weight‐bearing was suggested for 4 to 6 weeks with the use of two crutches, followed by progressive increase to obtain full weight‐bearing at 3 months.Study data collection, as well as radiographic and clinical evaluation, was performed at our institution by three external and independent investigators, who were not involved in the patients' treatment. Patients' characteristics  class to globally estimate surgical risk, and smoking habits) and treatment characteristics  were collected by evaluating hospital charts, clinical notes, and surgery reports.Radiographic data were obtained by reading the computerized images available in the X‐ray database computer system (MedStation program) of our institute. Standard radiographs were routinely performed preoperatively, immediately postoperatively, and during the outcome patient examinations 1, 3, 6, 12, and 24 months after surgery, according to our standardized follow‐up protocol. All radiographic evaluations were performed using a diagnostic liquid crystal display (LCD) CORONIS 5MP display  as a viewing monitor to identify fracture patterns and analyze the fractures and their outcomes. The fracture morphology was accurately evaluated and classified into different patterns based on preoperative radiographs and computed tomography (CT) scans . In Grade 0, no signs of OA are present, while in Grade 4, there are large osteophytes, as well as marked narrowing joint space, severe sclerosis, and definite bony deformity.Finally, examination of lateral and anteroposterior view radiographs at different follow‐up points, showing complete bridging bone/callus formation and the absence of radiolucent lines, was used to define bone healing.To measure the functional assessment of patients at last follow up, phone contact was attempted for all subjects included in the study, and a follow‐up appointment was fixed. Patients who accepted the follow up were evaluated and clinical results were measured with validated questionnaires . The visit consisted in a full examination of the lower extremities and gait of all patients.The knee examination included measurement of the ROM of the injured knee, varus‐valgus, and anterior–posterior stability, the presence of a flexion contracture, extension lag, and alignment. The ROM was measured as the passive arc of movement of the examined knee. Measurements were taken with each subject in supine position with the hip flexed on an examination table using a long‐arm goniometer with 50‐cm arms and central articulation. For healthy adult subjects, the normal value of ROM is between 0° and approximately 140° of knee flexion.The VAS is a validated, subjective measure for acute and chronic pain. Scores are recorded by making a handwritten mark on a 10‐cm line that represents a continuum between “no pain” and “extreme pain”The SF‐36 questionnaireThe SF‐36 measures eight scales, which contribute in different proportions to the scoring of both PCS and MCS measures. Each scale is scored separately from 0 to 100 points, where lower scores indicate poorer function. These scales are physical function, physical role, bodily pain, general health, vitality, social function, emotional role, and mental healthThe American Knee Society Score (AKSS)The knee injury and osteoarthritis outcome score (KOOS)Standardized answer options are used (5 Likert boxes) and all items have five possible answer options scored from 0 (no knee problems) to 4 (severe problems); each of the five scores is calculated as the sum of the items included. Scores are transformed between 0% and 100% using a precise algorithm Finally, a general questionnaire was administered to patients to obtain relevant information for the outcomes of the fracture, such as work and sports activities at the age of the trauma, and their resumption.2 or Fisher tests were applied to compare categorical variables between groups. The t‐test was applied to compare continuous variables between groups. P‐values were adjusted for age as a confounder with logistic regression for radiographic outcomes comparing the groups based on the type of fracture. P‐values were also adjusted for age as a confounder with ANCOVA for clinical outcomes, again comparing the fracture groups. Univariate analysis to compare clinical with radiographic outcomes was performed with a t‐test. Logistic regression analyses were carried out to identify predictors of the three major radiographic outcomes . Given the small sample size employed, an iterative strategy was used to properly choose the variables to retain in the regression models. An initial cut‐off of 0.25 was adopted, because more restrictive cut‐off points, including the classical threshold of 0.05, could fail in identifying important predictorsP < 0.05 .Statistical analyses were performed by an independent statistician from our university, using SAS 9.2  for Windows. Categorical variables were reported as number of patients and percentage. Continuous data were checked for a normal distribution with the Shapiro–Wilk test and expressed with mean and standard deviation or median and minimal–maximal values. χ2, one‐third of the patients were active smokers (33.33%), 7 patients (15.56%) had a diagnosis of hypertension, and 9 participants suffered from heart disease (20%) . According to the ASA classification of anesthesiological risk, 26 patients were classified as ASA 1 (57.78%), 15 (33.9%) as ASA 2 and 4 (8.89%) as ASA 3.Over a period of 5 years, 55 intra‐articular TPF treated with the ORIF technique in 55 patients fulfilling the inclusion criteria were identified. Among these, we could not evaluate 10 patients: 6 refused to participate, and a follow‐up address could not be retrieved for the other 4 patients . All fractures healed within 3 months following surgery. No significant differences were observed between 41‐B and 41‐C fractures comparing patients' demographic and clinical aspects (Table P = 0.040).In more than half of the patients (66.67%), AO 41‐B fractures  were reported, while the remaining 15 had AO 41‐C fractures (complete articular fractures). The sub‐type AO 41‐B3 was the most common and was reported in 62.22% of patients. In almost all patients (88.89%), high energy trauma was the most common type of injury; traffic accidents were reported in 33 cases (73.33%), followed by falls (15.56%) and sports‐related injuries (11.11%). There was no correlation between AO 41‐B or 41‐C fractures and type of trauma. Concomitant fractures were found in 18 injured patients (40%). Time between trauma and surgery ranged from 1 to 12 days, with a median time of 1 day. The median duration of hospitalization was 7 days . In the present series, 18 patients (40%) were reported to have soft tissue damage. However, only 3 cases (7.67%) presented with associated open fractures: 2 Gustilo grade I and 1 Gustilo grade II. Six cases (13.33%) of TPF were previously stabilized with an axial external fixator (2‐step surgery) before treatment with ORIF techniques (3 open fractures and 3 severe soft tissue damage). AO 41‐C TPF were more frequently stabilized with external fixation with respect to 41‐B (In 24 (53.33%) patients, postoperative radiographs (12 months after surgery) showed anatomical reductions .Because development of PTOA has been reported after TPF, postoperative radiographs 24 months after surgery were analyzed according to the Kellgren–Lawrence scale (Table The mean ROM was 117.4° (±10.0°). The VAS reported an average score of 2.9 (±2.2). Mean SF‐36 PCS was 41.4 (±11.2) and mean MCS for mental status was 47.4 (±11.5). These results are summarized in Table The AKSS  mean score was 79.0 (±16.1), with an overall good outcome. The result of AKSS was “excellent” (score > 80) in 23 patients (51%), “good” (score 70–79) in 5 (11%), “fair” (score 60–69) in 10 (22%), and “poor” (< 60) in 7 (16%). The mean KOOS overall score was 69.0 (±20.1). The mean KOOS was 82.1 (±16.2) for the pain subscale, 77.2 (±22.2) for symptoms, 82.0 (±18.5) for daily function, 47.1 (±33.5) for sports and recreation, and 55.8 (±28.6) for QoL.P = 0.012). Moreover, AKSS and KOOS QoL were higher for the AO 41‐B fractures compared to AO 41‐C fractures . In contrast, SF‐36 PCS was higher for 41‐C fractures compared to 41‐B fractures (P = 0.0198). No other statistical differences were found evaluating the clinical outcomes between the two groups.After adjustment for age as a confounder, reduced ROM was observed in 41‐C fractures compared to 41‐B fractures (P = 0.019), higher pain measured by VAS (P = 0.039), lower clinical scores AKSS (P = 0.0006), lower KOOS pain (P = 0.001), lower KOOS symptoms (P = 0.0083), lower KOOS function (P = 0.0008), lower KOOS sports/recreation (P = 0.0017), lower KOSS QoL (P = 0.0005), and lower SF‐36 PCS (P = 0.0002).Among the 37 patients that were employed at the time of injury, 24 patients (65%) returned to full work capacity. A total of 13 patients (35%) did not return to work or needed to change jobs because of limitation caused by their injury. These patients, compared to those who returned to previous work, showed lower ROM (P = 0.02), lower AKSS (P = 0.0026), lower KOOS pain (P = 0.03), lower KOOS symptoms (P = 0.04), lower KOOS function (P = 0.02), lower KOOS sports/recreation (P = 0.035), lower KOOS QoL (P = 0.0674) and lower SF‐36 PCS (P = 0.001).In addition, 16 patients (64%) did not return to their previous level of sports activity because of pain. These patients showed higher VAS pain (P = 0.470) and a better outcome in daily life activities (KOOS ADL P = 0.0488) and knee function , and less pain (KOOS pain P = 0.0105) compared to patients with a gap or step‐off >1 mm  . Weak negative correlations were observed between BMI and all subscales of KOOS  and weak to moderate negative correlations were identified between age and KOOS subscales. Finally, we identified a significant relationship between the presence of PTOA and clinical outcomes at last follow up. In particular, a worse clinical outcome was reported for patients with PTOA compared to patients without PTOA, as demonstrated by a statistical difference in ROM (P = 0.0053), AKSS (P = 0.021), KOOS sport (P = 0.0167), KOOS QoL (P = 0.0227), and SF‐36 PCS (P = 0.0142), and SF‐36 MCS (P = 0.0381)   , resolved with 6 months of therapy, and there were 4 cases of transitory deficit of the common peroneal nerve (CPN), which spontaneously resolved in 3 months.The management of articular TPF remains an important issue for orthopaedic surgeons because of complex fracture geometry and the fractures' often unsatisfactory outcomes and high complication rateWe enrolled 45 patients in our study, with a mean age of 54.5 years and mean follow‐up time of 57.18 months. More than half of the patients (66.67%) had partial articular fractures (AO 41‐B), while the remaining subjects (33.33%) had complete articular fractures (AO 41‐C). The mean KOOS score was 69.0, while the mean AKSS and SF‐36 PCS scores were, respectively, 79.0 and 41.4. Significant correlation between age and functional results  and between BMI and KOOS Pain, ADL, Sport, and QoL were found. Complete articular TPF (AO 41‐C) tend to develop PTOA more frequently with respect to partial articular TFP, so the first fracture type had worse clinical outcomes.Patients with postoperative non‐anatomical reduction with step‐off >1mm and tibial malalignment experienced more pain. Moreover, the presence of PTOA at 2 years follow‐up was predictive of worse AKSS scores.Our case series was mainly represented by middle‐aged patients with high‐energy trauma, most of whom were victims of traffic accidents. Although in the past few decades, treatment strategies for high‐energy fractures of the tibial plateau have changed, the traditional ORIF using locking plates with or without previous external fixation allowed us to obtain anatomical and postoperative optimal alignment in half of the patients at last follow‐up.et al.However, even if an anatomical reduction of the fractures is mandatory , even if Van Dreumel et al. included in their analysis simpler fractures treated only by screwset al. and Volpin et al. found that knee stability related to better clinical outcomes rather than the quality of reduction and residual articular step‐off In our series, even if worsening fracture types resulted in worse outcomes, there was no statistical correlation between radiographic outcomes and fracture patterns. Hence, according to Singleton et al. did not find any relationship between radiological OA 1 year after surgery and clinical outcomes because PTOA is a slow process that requires more than 1 year to develop. For this reason, we performed its evaluation at 2 years after surgery and found that it is a significant prognostic factor of lower AKSS score, but it is not a significant predictor of lower KOOS or SF‐36 score. This result is probably due to the difference among questionnaires  and because more time is necessary for the development of a symptomatic osteoarthritic knee. According to our data, half of our patients developed signs of PTOA 2 years after surgery. These patients had worse clinical‐functional outcomes. However, we could not exclude that early OA signs were present before the TPF in adult subjects, but, according to Marsh et al., the initial injury can be the most important factor leading to PTOA, despite accurate reductionet al. suggested that TKR should be considered in cases of complex articular fractures of the proximal tibia in elderly patients with symptomatic OA or with poor bone quality that makes internal fixation hazardousThe incidence and onset of PTOA after TPF are reported in the literature with a wide range, between 17% to 83% in comminuted fractureset al.et al. et al. and Jansen et al., even for the more complex AO 41‐C fractures with low complication rateet al. did not find a direct correlation between age and KOOS scoreNumerous tests for functional outcome after knee injuries have been described in the literature. According to Manidakis et al. reported that one‐third of patients with surgically treated TPF did not return to work after injury, and approximately 50% did not return to their previous sports levelFinally, TPF have a significant socioeconomic impact, mainly due to time taken off workAlthough our study deals with a relevant topic in trauma surgery, several potential limitations may have influenced its results, as it was retrospective and a single‐center investigation. There was possible bias due to the different number of 41‐B and 41‐C TPF included, the varied skill of the different surgeons performing the operations , the relatively limited sample size , and the presence of early OA radiographic signs at the time of trauma (even if our series was mainly represented by middle‐aged patients). Moreover, X‐rays were used to evaluate radiographic outcomes. In fact, the measurement of articular step‐off was also performed using knee postoperative X‐rays in other studies Despite these limitations, our study reported detailed radiographic outcomes at 12‐month and 24‐month follow up, and mid‐term clinical functional outcomes of a consecutive series of patients affected by articular TPF and surgically treated by ORIF. Furthermore, as the development of PTOA in half of our cases was already observed 2 years after trauma, we strongly believe that patients with joint malalignment and/or unsatisfactory anatomical reduction should have a longer follow up, with an associated long‐term rehabilitative program and a chondroprotective therapy to prevent PTOA.In conclusion, the present study first provides useful data regarding the importance of postoperative radiographic and functional follow up after TPF. Second, it highlights that the early radiographic features  may be predictive for clinical outcomes in terms of pain felt by patients at mid‐term follow up, and PTOA development may be predictive for knee function measured with AKSS. Finally, it confirms that the challenging management of TPF consists not only in the mere treatment of acute knee damage but also in the effort to limit the consequent clinical, social, economic, and health impact of these severe injuries.Table S1. Demographic data, fracture type, radiological, and clinical outcome scores of our case series.Click here for additional data file.


A hallmark of a perceptual expert is the ability to detect and categorize stimuli in their domain of expertise after brief exposure. For example, expert radiologists can differentiate between “abnormal” and “normal” mammograms after a 250 ms exposure. It has been speculated that rapid detection depends on a global analysis referred to as holistic perception. Holistic processing in radiology seems similar to holistic perception in which a stimulus like a face is perceived as an integrated whole, not in terms of its individual features. Holistic processing is typically subject to inversion effects in which the inverted image is harder to process/recognize. Is radiological perception similarly subject to inversion effects? Eleven experienced radiologists  and ten resident radiologists  judged upright and inverted bilateral mammograms as “normal” or “abnormal”. For comparison, the same participants judged whether upright and inverted faces were “happy” or “neutral”. We obtained the expected inversion effect for faces. Expression discrimination was superior for upright faces. For mammograms, experienced radiologists exhibited a similar inversion effect, showing higher accuracy for upright than for inverted mammograms. Less experienced radiology residents performed more poorly than experienced radiologists and demonstrated no inversion effect with mammograms. These results suggest that the ability to discriminate normal from abnormal mammograms is a form of learned, holistic processing. Breast cancer is the most prevalent cancer in women, with approximately 250,000 new cases reported each year. The ability to accurately diagnose breast cancer in an X-ray image is an essential medical skill for early detection and treatment. However, identifying breast cancer in an X-ray is not an easy perceptual task and requires many years of experience and practice. One of the key differences between an expert and novice radiologist is their ability to detect abnormalities within a medical image in the blink of an eye. It has been speculated that holistic processing (widely reported in face recognition) helps enable this rapid detection. In the present study, we test this claim by employing an inversion paradigm, which has been demonstrated to disrupt holistic processing. Resident and experienced radiologists were asked to identify abnormal and normal mammograms presented in their upright and inverted orientations. We found that experienced radiologists were more accurate at identifying abnormalities in upright mammograms than in inverted mammograms. By comparison, the radiology residents performed more poorly than the experienced radiologists overall and their performance was not affected by inversion. The expert inversion effect indicates that experienced radiologists employ holistic processes to assist their rapid detection of breast cancer and these processes are disrupted when the mammogram is turned upside down. Our results suggest that teaching methods in medical imaging may benefit by including a holistic approach in which students are trained in the rapid detection of many mammogram exemplars.A hallmark of the human visual system is our ability to make rapid visual categorizations in fractions of a second, whether we are interpreting the meaning of a picture  while ignoring information from the other half   of briefly presented upright and inverted faces. Recognition of facial expressions, like facial identity, recruits holistic perception that is disrupted by inversion , average 18 years in practice (range 6 to 37 years) reading, on average, 6045 cases  in the last year. The other ten participants were radiology residents who had fewer than 5 years of experience , average 3 years in practice (range 2 to 5 years) reading, on average, 297 cases (range 20 to 500) in the last year. The expertise cut-off was based on previous studies  views or craniocaudal (CC) views of bilateral breasts Fig. . Half ofhttp://www.fantamorph.com). At least 50 control points were placed on each face, and control points were added to remove obvious artifacts in the resulting morph. Hair and clothing information was removed with Adobe PhotoshopTM graphics program . Faces were scaled to fit within a frame of 250 × 375 pixels and pasted on a black background  and one block of 40 mammogram trials . Each trial began with a fixation cross presented at the center of the screen (500 ms), followed by the target mammogram or face stimulus, a noise mask (1000 ms), followed by a response screen Fig. . In the d’), Experience  was a between-group factor and Orientation  was a within-group factor. A main effect of Orientation was statistically significant  = 36.73, p < 0.0002, ηp2 = 0.53), displaying the classic face inversion effect where expression classification was impaired in the inverted orientation relative to the upright orientation. With face sensitivity, no Experience effect was found  = 0.1, p > 0.76), nor was there an interaction between Experience and Orientation . No Experience effect was found  = 0.86, p > 0.37, ηp2 = 0.04).A 2 × 2 mixed ANOVA was conducted for the face sensitivity , there was a main effect of Experience  = 6.18, p < 0.02, ηp2 = 0.22; experts are better). A direct comparison using paired t-tests revealed that experts had better performance for upright mammograms (t(10) = 3.6, p = 0.0019, Cohen’s d = 0.45) compared to residents. There was no reliable difference for the inverted mammograms (p > 0.2). A Group by Orientation interaction was obtained  = 11.91, p = 0.003, ηp2 = 0.09), reflecting the presence of an inversion effect for the experts but not residents. The inversion effect was assessed with paired t-tests. These reveal a significant inversion effect for experts (t(10) = − 2.8, p = 0.018, Cohen’s d = − 0.51). The effect for residents was not significant and, in any case, goes in the opposite direction from what would be expected (p > 0.33)  or Orientation  = 2.89, p > 0.11, ηp2 = 0.04) effects.For the ANOVA on the mammogram data, Experience  was a between-group factor and Orientation  was a within-group factor. For mammogram recognition sensitivity (33) Fig. . Reactiot(19) = − 0.58, p = 0.56, Cohen’s d = − 0.13). However, the experienced radiologists were less likely to misclassify a normal mammogram as abnormal  = − 4.74, p < 0.01, Cohen’s d = − 1.05). This means that the criterion of experienced radiologists was more strongly biased toward a “normal” classification than less experienced radiologists (t(19) = 2.73, p = 0.008, Cohen’s d = 0.6). There were no significant differences in hit rates, false alarms, or biases between experts and residents for the inverted mammograms and upright and inverted faces. These results are tabulated in Table Further, we explored the differences in mammogram performance, comparing “hit” and “false alarm” rates. Experienced and resident radiologists did not differ in their ability to classify an abnormal mammogram as “abnormal” (hit rates) in upright mammograms  = 15.2, p < 0.001, R2 = 0.45, 95% CI = 0.018, 06). However, years of experience was not related to the ability to discriminate inverted mammograms  = 2.179, p < 0.16, R2 = 0.1, 95% CI = − 0.006, 0.03; Fig. Figure F = 8.49, p < 0.009, R2 = .30, 95% CI = 0.018, .059; Fig. There is also a significant correlation of the magnitude of the inversion effect (difference between upright and inverted mammogram discrimination) with years of experience  and residents (< 5 years of experience) were asked to classify upright and inverted mammograms as either normal or abnormal. As comparison stimulus, the same participants judged whether upright and inverted faces displayed a happy or neutral expression. For faces, both resident and experienced radiologists exhibited the classic face inversion effect where face expression discrimination was better in the upright orientation than the inverted orientation. For mammograms, experienced radiologists showed superior discrimination relative to resident radiologists for mammograms presented in their upright orientation. Although the overall performance of resident radiologists was worse than the experienced radiologists, their detection rates were unaffected by orientation and were essentially the same for upright and inverted mammograms. When the mammograms were inverted, the discrimination scores of the experienced radiologists dropped to the same level as demonstrated by resident radiologists. These results are consistent with previous studies of perceptual expertise showing that, with extensive domain-specific experience, experts access holistic information in an upright stimulus, but holistic information is impaired when the stimulus is inverted  was comparable to detection levels of expert radiologists in previous studies for upright mammograms (d’ = 1.14) (experiment 1; Evans, Haygood, Cooper, Culpan, & Wolfe, d’ = 1.23 for the intact stimulus compared to d’ = 1.09 for the scrambled stimulus.Although inversion impaired mammogram detection for the experienced radiologists, it did not completely abolish the expertise advantage. The performance of experienced radiologists in this study (The holistic account of mammogram expertise is consistent with eye-tracking studies which show that, in comparison to non-experts, experts typically perform domain-related tasks with fewer fixations, longer saccades, and less coverage of the image (Krupinski, The emergence of a holistic strategy with radiological experience has implications for training medical students. In our study, the expert radiologists acquired holistic strategies implicitly over many years of clinical experience, evaluating thousands, even tens of thousands of mammograms images. It is interesting to speculate whether this perceptual knowledge can be taught explicitly during medical training by presenting medical students with many images of abnormal and normal mammograms, asking them to judge the normality of each image and providing the appropriate feedback. A perceptual expertise protocol might accelerate the learning process and allow radiological trainees to achieve expert performance more quickly. The perceptual expertise training approach has been successfully applied in other medical domains that require visual diagnosis. For example, participants showed reliable gains in their ability to detect melanoma skin lesions after four 30-min sessions of perceptual expertise training (Xu, Rourke, Robinson, & Tanaka, The goal of this study was to measure the holistic processing of expert mammographers by employing the inversion test—a standard measure of holistic processing used in the face recognition research. The main finding was that experienced radiologists exhibited a robust inversion effect as evidenced by their better discrimination of upright mammograms than inverted mammograms. In contrast, the less experienced, resident radiologists performed more poorly than experienced radiologists and their discriminations were the same for upright and inverted mammograms. Critically, detection performance improved and the inversion effect increased for radiologists who had more experience diagnosing mammogram images, suggesting that holistic abilities developed as a function of perceptual experience. Our results have implications for training medical students by emphasizing the role of experience and the learning principles of perceptual expertise.


Atriplex lasiantha Boiss.Plants represent an intricate and innovative source for the discovery of novel therapeutic remedies for the management of various ailments. The current study has been aimed to validate the therapeutic potential of ethnomedicinally significant plant The polarity based extraction process was carried out using fourteen solvents to figure out best extraction solvent and bioactive fractions. Total phenolic-flavonoids contents were quantified colorimetrically and polyphenolics were measured using HPLC-DAD analysis. Moreover, the test samples were tested against several diseases targets following various assays including free radicals scavenging, antibacterial, antifungal, cytotoxic and antileishmanial assay.A. lasiantha. Significant quantity of rutin i.e., 0.3 μg/mg was quantified by HPLC analysis. The methanol-ethyl acetate extract of A. lasiantha exhibited maximum total antioxidant and total reducing power with 64.8 ± 1.16 AAE/mg extract respectively, while showing 59.8 ± 1.07% free radical scavenging potential. A significant antibacterial potential was exhibited by acetone-distilled water extract of A. lasiantha with 11 ± 0.65 mm zone of inhibition against B. subtilis. Considerable antifungal activity was exhibited by ethyl acetate-n-hexane extract of aerial part of A. lasiantha with 14 ± 1.94 mm zone of inhibition against A. fumigatus. Highest percentage of α-amylase inhibition (41.8 ± 1.09%) was observed in ethyl acetate-n-hexane extract. Methanol-acetone extract of A.lasiantha demonstrated significant inhibition of hyphae formation with 11 ± 0.49 mm bald zone of inhibition. Significant in-vitro cytotoxicity against Hep G2 cell line has been exhibited by methanol-chloroforms extract of A.lasiantha.Among the solvent fractions, maximum yield was obtained with methanol-water extract i.e., 11 ± 0.49%. Maximum quantity of gallic acid equivalent phenolic content and quercetin equivalent flavonoid content were quantified in methanol-ethyl acetate extract of Atriplex lasiantha Boiss. for the discovery of biologically active compounds through bioassay guided isolation against various diseases.The current study reveals the prospective potential of  Natural resources represent an important reservoir of bioactive compounds for the development of novel drug entities –3. AmongAtriplex encompassing about 300 species of angiosperms is well recognized ethnomedicinal genus which exists widely and utilized by aboriginal folks [Atriplex nummularia and Atriples leucoclada possess anti ulcerative, anticolitis, antifungal and mollusicidal activity [Atriplex farinose possesses antifertility potential, it also has anti ulcerative and anticolitis activity [Atriplex conferitifolia is said to possess a noteworthy activity against cervical cancer cells of human and have analogous potential to a cancer drug called “Onxol” which is FDA approved [Atriplex vesicaria show antileukemic activity [Atriplex glauca exhibit cytotoxicity against human colon cancer cells [Atriplex semibaccata, Atriplex portulacoides, Atriplex parvifolia and Atriplex inflate have been reported to have insecticidal activity against Tribolium spp. [Atriplex canescens are used to cure infections of gastrointestinal in Mexico and its methanolic extract show anti-bacterial activity against E.coli and Salmonella typhimurium [Atriplex crossifolia are used in the treatment of yellow jaundice and throat infection [Henceforth, nowadays there has been an incredible surge in ethnomedicinal herbal medications , 11. Theal folks , 13. It activity , 15. Atractivity . Atripleapproved . It has activity , whereaser cells . Atripleium spp. . Leaves himurium . In Pakinfection .Atriplex lasiantha of genus Atriplex. A. lasiantha Boiss. (Orache) is an annual erect branched herb, having deep spreading roots. It is distributed in wet mountains, deserts, arid semi-arid mountains. It is also found in the plains of Indus and Balochistan and is mainly used for food and forage. The current study compiles an exclusive report on antioxidant, antibacterial, antifungal, antidiabetic, antileishmanial and cytotoxic activity of Atriplex lasiantha Boiss. by using multimode polarity extraction system.Presence of diverse secondary metabolites and their medicinal importance motivated us to explore the species In this study, solvents that were used for extraction purpose were of analytical grade. The solvents included dimethyl sulfoxide, methanol, acetone, ethyl acetate, ethanol, chloroform, and n-hexane. The solvents and reagents like dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferric chloride, trichloroacetic acid, ammonium molybdate, potassium ferricyanide, sulphuric acid, aluminium chloride and potassium acetate were purchased from Merck, Darmstadt, Germany. 2,2-diphenyl-1-picryhydrazyl (DPPH) and Folin-Ciocalteu reagent were bought from Sigma–Aldrich . Tween-20 was bought from Merck , while Phosphate Buffer Saline (PBS), nutrient agar, Sabouraud dextrose agar (SDA), Ttryptone Soy Broth (TSB),sterile normal saline solution (0.9%), sea salt, 0.5% triton x-100, roxithromycin and cefixime, clotrimazole and amphotericin B, doxorubicin, vincristine, surfactin, were purchased from Sigma , Medium ISP4 (was Prepared in lab), and those reagents that were used i.e.myricetin, gallic acid, rutin, kaempferol catechin,caffeic acid, quercetin, ascorbic acid, were also purchased from Merck, Darmstadt, Germany.Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 10240) and Bacillus thuringiensis (ATCC 6633) and Gram negative strains: Klebsiella pneumonia (ATCC 1705), and Escherichia coli (ATCC 25922). Whereas,fungal strains that were used as test organism were Fusarium solani (FCBP 0291), Aspergillus flavus (FCBP 0064), Aspergillus fumigates (FCBP 66), Mucor species (FCBP 0300) and Aspergillus niger (FCBP 0198). HepG2 cancer cell line (RBRC-RCB1648), Leishmania tropica (kMH 23 strain) and Streptomyces (85Estrain) were also used.The bacterial strains used in biological assays were Gram positive strains: A. lasiantha was collected from Peshawar, Khyber Pakhtunkhwa (KP) Pakistan and identified and verified by Prof. Dr. Mushtaq Ahmad, Department of Plant sciences, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad. Plant was selected on the basis of its ethnopharmacological significance, safety profile in humans, cost effectiveness, ease of availability and was deposited in MoSAEL Lab’s herbarium, Quaid-i-Azam University Islamabad, Pakistan (voucher number MOSEAL-347). Aerial part of A. lasiantha was apportioned from any deteriorated or foreign matter. Afterwards, they were dried in well-ventilated area at room temperature for four weeks and coarse grinded via cutter machine [The sample of  machine , 24.n-hexane: ethyl acetate, EN: ethanol: n-hexane, MEC: methanol: chloroform MEEA: Methanol-Ethyl acetate, MEA: methanol: acetone, AW: acetone: water, MED: methanol: water. Fourteen different solvents of wide ranging polarity were used, includes seven single solvent and seven in combinations (1:1). Plant powder (30 g) was socked in 120 mL of respective solvents for maceration in separate Erlenmeyer flasks and left for 3 days at room temperature. Shaking was done on daily basis followed by ultrasonication at a frequency of 40 KHz. After 3 days, filtration was done by Whatman No.1 filter paper and next same volume was added. Second volume of respective solvents was filtered after two days and third volume of solvent was added. Third volume of solvent was again filtered after two days, and all filtrate was placed at room temperature for drying. The filtrates were vaporized accordingly and the dried plant extracts were then stored in pre-weighed labeled vials at − 20 °C for further analysis [Initially, the plant material was washed with water to remove dirt particles and then shade dried for four weeks at room temperature. After that aerial part was grinded by using electric knife mill and then stored in tight jar. The powered plant material was subjected to extraction followed by sonication and maceration with analytical grade solvents i.e., N: n-hexane, C: Chloroform, EA: Ethyl acetate, A: Acetone, E: Ethanol, ME: Methanol, W: Distilled water, NEA: analysis .The percentage yield of dried crude extracts recovery after extraction was evaluated gravimetrically by using this formula which are as follows.Where “a” and “b” corresponds to the weight of dried extract and weight of plant powder used respectively.2CO3. Subsequently, the reaction mixture was incubated for 30 min at 37 °C. The absorbance of reaction mixture was recorded at 630 nm with the help of microplate reader . The standardization curve  of gallic acid was used for quantification of phenolic content. Resulting phenolic content was expressed in term of gallic acid equivalent (μg GAE/mg extract).Total Phenolic Content present in plant sample was calculated by using already reported spectrophotometric method . Briefly3, 10 μL of 1 M KCH3CO2 and 160 μL of H2O in 96 well plate. Incubated for about 30 min after that absorbance of reaction mixture was recorded at 415 nm with the help of microplate reader . The standardization curve  of quercetin was used for quantification of flavonoid content. Resulting flavonoid content was expressed in term of quercetin equivalent (μg QE/mg extract).Total Flavonoid Content present in plant sample was calculated by using already reported method which was based on formation of aluminum chloride complex . Test saA. lasiantha extract for polyphenols were carried out by following already reported method [−1methanol and standards i.e.Gallic acid, rutin, quercetin, catechin, caffeic acid, apigenin, myricetin and kaempferol were prepared in methanol and then diluted to get final concentration of 10, 20, 50, 100, 200 μg/ml respectively with the help of sonication in ultrasonic water bath. The gradients system utilized was comprised of solvent A  and solvent B . The flow rate was kept at 1 mL/min. Each sample solution (20 μL) was loaded into column. Before the injection, each sample was filtered with 0.2 μm membrane filter (sartolon polyamide). The gradient volume of B was utilized as follows: 0–50% in 0–20 min, 50–100% in 20–25 min and then 100% from 25 to 30 min. The absorption of samples was recorded at different wavelengths e.g., gallic acid (257 nm) catechin (279 nm), rutin (257 nm), caffeic acid (325 nm) and kaempferol, quercetin, myricetin (368 nm). The reconditioning of column was done for 10 min before each analysis. By using the standard, integration of peak has been done for quantification of polyphenols. All chromatography was processed at an ambient temperature.For interpretation of significant manifestation of phenolics and good biological activity, we selected MEEA extract for HPLC-DAD analysis. Chromatographic analysis of d method . In thisA. lasiantha extracts were estimated by measuring the change in 2, 2-diphenyl-1-picryhydrazyl (purple colored) to diphenyl picrylhydrazine (Yellow Colored). The spectrophotometric method was used for investigation [Radical scavenging activity of tigation , 30. As S”and “AbC”correspond to absorbance of sample and control respectively.Where the “Ab2SO4, 4 mM (NH4)6 MO7O24.4H2O and 28 mM NaPO4.100 μL aliquot of each sample was mixed with 1 mL solution of the reagent. After that the reaction mixture was incubated at 90 °C for 95 min. Ascorbic acid and DMSO were used as positive control and as negative control respectively The absorbance was recorded at 645 nm with the aid of microplate reader  and was expressed in term of ascorbic acid equivalent per microgram extract (μg AAE/mg extract).Total antioxidant capacity was estimated by phosphomolybdenum method . Reagent2HCl3O2 was added and resultant mixture was centrifuged at 3000 rpm for 10 min. Lastly, the supernatant was mixed with 0.5 mL FeCl3 followed by 0.1 mL distilled water. The absorbance of reaction concoction was recorded at 700 nm with the aid of microplate reader  and was expressed in term of ascorbic acid equivalent per microgram extract (μg AAE/mg extract).The reducing capacity of plant extracts was assessed by formerlydefined method  with smaS. aureus (ATCC 6538), Micrococcus luteus (ATCC 10240) and Bacillus thuringiensis (ATCC 6633) and Gram negative strains: Klebsiella pneumonia (ATCC 1705), and Escherichia coli (ATCC 25922). Sterile loopful full of spores was inoculated in 10 mL aliquot of nutrient broth followed by incubation at 37 °C for 24 h. After that, turbidity of each inoculum was maintained at 104 CFU/mL by comparing it with 0.5 McFarland turbidity BaSO4 standards. A volume of microbial preparation (100 μL) was spread on agar plates with subsequent addition of 100 μg of sample to the plates and incubated for 24 h at 37 °C. After this clear zone of inhibition around the discs was recorded with vernier caliper to the nearest. Cefixime and DMSO were used as positive and negative control respectively.Disc diffusion technique was used for investigation of bacterial susceptibility of plant extract as previously reported , 34. FivFusarium solani (FCBP 0291), Aspergillus flavus (FCBP 0064), Aspergillus fumigatus (FCBP #66), Mucor species (FCBP #0300) and Aspergillus niger (FCBP 0198). Initially fungal spores were refreshed by swabbing on sterile SDA plates followed by incubation for 5–7 days at 28 °C. After this, spores of fungal strains were harvested in 0.02% Tween-20 solution and their turbidity was maintained at 104 CFU/mL by comparing it with 0.5 McFarland turbidity BaSO4 standards. A suspension (100 μL) of test strain was spread on sterile SDA plate then 100 μg/ml of sample was poured on seeded plates followed by incubation for 28 °C for 24–48 h. After this clear zone of inhibition around the discs was recorded with Vernier caliper to the nearest. Clotrimazole and DMSO were used as a positive and negative control respectively.Disc diffusion method was used with for investigation for the fungus susceptibility of plant extract , 36. FivSelected samples (≥10 mm zone of inhibition) were further evaluated for minimum concentrations (MIC) by using standardized 3- fold micro dilution method. Bacterial suspension (50 ml) of each tested bacteria were grown in broth and then added in 96 well plate with different concentrations if the test samples (100 μg/ml to 7.40 μg/ml). The final volume in the well was kept 200 μL. After the incubation period, plates were analyzed by ELISA Spectrophotometer to determine the growth of bacteria in each respected well. The minimum concentration of each sample that was showing no growth are taken as MIC (Minimum inhibitory concentration).For determination of amylase inhibition spectrophotometric method was used with that was previously described . PhosphaS”, “AbN”and “AbB”corresponds to absorbance of sample, negative control and blank respectively.Whereas, “AbHemolytic activity was carried out by using fresh isolated blood from human body. For isolation of RBCs, 1 mL fresh blood was centrifuged at 14, 000 rpm for 5 min. After that, 200 μL from it was mixed up with PBS (pH = 7.2) for preparation of erythrocyte suspension. Test sample (100 μL) was added in eppendorf containing 100 μL erythrocytes suspension followed by incubation for 60 min at 35 °C. After incubation, the reaction mixture was again centrifuged at 10,000 rpm/10 min.The absorbance was recorded at 530 nm with the help of microplate reader  to find out the percent hemoglobin release. Triton X-100 and DMSO were used as positive and negative control respectively. All experiments were repeated three times and results were expressed in term of % Hemolysis per mg of plant extract. Hemolysis was find out by using the following formula:S”, “AbNC”and “AbPC”corresponds to absorbance of sample, negative control and positive control respectively.Whereas, “AbStreptomyces (85E strain) cultured in minimal ISP4 media was used for this assay [Streptomyces. Surfactin and DMSO was used as positive and negative control respectively.is assay . Test sa2 humified incubator. The reaction was stopped with the addition of 50 μL of cold 20% TCA and then again incubated for 60 min at 4 °C for cell fixation. Subsequently, the cells become immobilized, washed with tap water four times followed by staining with 50 μL of 0.057% SRB in 1% acetic acid at room temperature. Wells were again placed overnight for drying and washed four times with 1% acetic acid. Subsequently, 0.2 mL test sample of 10 mM Tris base (pH 10) was then added and left for one hour to solubilise the bounded dye. Doxorubicin and DMSO were used as positive and negative control respectively. In order to evaluate the percentage of cells survival, microplate reader  was used at 515 nm for measuring the optical density. Percentage of cell growth inhibition was determined by using formula:The in-vitro cytotoxic activity against Hep G2 cancer cell line (RBRC-RCB1648) was evaluated with the help of standard protocol , 39. TesWhere “OD” corresponds to optical density. 0 day control was conceded by adding same number of cells followed by incubation at 37 °C for 1 h and followed the same protocol.Leishmania tropica (KMH 23 strain) culture supplemented with bovine fetal serum. Density of culture was maintained at 1 × 106 promastigotes per mL. Test sample (20 μL) at final concentration of 100 μg/ml was added to 180 μL of culture followed by incubation at 24 °C for 72 h in 5% CO2 humified incubator. The absorbance was recorded at 540 nm with the assistance of microplate reader . The lived promastigotes were counted under inverted microscope with the help of hemocytometer and their LC50 was calculated by table curve (2D ver. 4 software). DMSO and Amphotericin B were used as negative and positive control respectively. Percentage mortality was calculated by using the formula:MTT colorimetric assay as described previously  was usedS” and “AbC”corresponds to absorbance of sample and absorbance of control respectively.Where “AbP < 0.05 were considered significant. The data was analyzed via Statistix 8.1 software, expressed as mean ± standard deviation. Correlation investigation was conceded out by using regression line in Microsoft Excel 2013 Program. IC50 and LC50 were calculated by table curve 2D version 4 software. Graphs were plotted by Origin 8.5 software.Multiple group comparison was performed by Two way ANOVA followed by Bonferroni post test by GraphPad Prism Version 5.0  in which the A. lasiantha were prepared in different solvents and investigated to explore the spectrum of phytochemicals and biological activities. Percentage recovery for different solvent extracts is summarized in Table w/w) followed by 10 and 7.7% for methanol-ethyl acetate and methanol extracts respectively. Whereas, n-hexane fraction yielded the least content (1.7%) because of being least polar among the solvents used.A total of 14 different extracts from the aerial parts of A. lasiantha followed by MW > AW > EAN > MEC > MEA > W > ME > EA > A > E > EN > C > N.The profile of total phenolic and flavonoid content was determined from standard curve calibration of gallic acid and quercetin respectively. The TPC and TFC has been provided in Fig. Fingerprinting the chemical profile of analytes via HPLC is considered as an unpretentious, reproducible, profound and reliable method . ConsideA. lasiantha. To establish the antioxidant nature of the different extracts of A. lasiantha, free radical scavenging, Total Antioxidant Capacity and Total Reducing Power assays were carried out as shown in Fig. Different mechanisms are involved such as free radical scavenging, prevention of chain initiation and peroxide decomposition. There is no single method which can offer a comprehensive, accurate and quantitative estimate of antioxidant efficiency and antiradical efficacy . Therefo2 = 0.9575). Consequently, it can be concluded that different polyphenols in A. lasiantha extracts are responsible for antioxidant activity.Among all extracts MEEA and MEW showed highest scavenging potential i.e. 59.8 ± 1.07% and 56.7 ± 1.08% respectively at 200 μg/ml Fig. . For othTotal antioxidant capability was estimated by phosphomolybdenum method based on formation on phosphate molybdate complex (green in color). The extracts reduced Mo (VI) to Mo (V) and give maximum absorbance at 695 nm. Methanol-ethyl acetate extract exhibited highest antioxidant capability i.e. 64.8 ± 1.16. The antioxidant have a strong correlation with the solvent that was used and a positive linear correlation (R2 = 0.8402) was demonstrated between TPC and antioxidant activity which is in strong agreement with earlier reported documentation of different authors .A. lasiantha was estimated by potassium ferricyanide method based on conversion of ferric chloride to ferrous chloride which results change in color from yellow to green. The intensity of appearance of green color is directly proportion to the sample reducing power [Reduction potential of ng power . Highestng power  and demoA. lasiantha reveals that the N, C and EA show almost the same activity against various bacterial strain and all the three samples are moderately active against all the test strains. An average of approximately 7 mm inhibitory zone has been exhibited by N, C and EA against K. pneumonia, M. luteus and B. subtilis, which is significantly small in comparison with the positive control i.e., Cefixime, which exhibited greater than 20 mm zone of inhibition against majority of the test strains as shown in the Table . Medicinal plants with therapeutic uses can be screened for the presence of antimicrobial entities. As a first step in investigating, the antimicrobial assays provides a glimpse of the microbio-cidal or microbio-static nature. In present study, the susceptibility of extracts was evaluated by agar disc diffusion method. The extracts exhibited varying degree of growth inhibition of the strains under investigation  is summarized in Table A. lasiantha was first time evaluated against Leishmania tropica. In case of aerial part of A. lasiantha, NEA, N and W extracts showed significant leishmanicidal potential with 50% mortality at 13.5 μg/mL, 12.8 μg/mL and 16.8 μg/mL respectively  inhibition is a popular strategy in the treatment of cancer. Plants have proved as a vital resource of anticancer compounds. Most of inhibitors are investigated for cancer but deregulation of kinase function is also involved in neurological, immunological, infectious and metabolic diseases. Researchers are diverted for development of kinase inhibitors, so cancer along with these disorders can be treated . There aferation . A greatferation . Signal feration , 57. Unuferation  and leadchanisms . Genome ptomyces . AfsR, Pptomyces . In the Leishmania which is transmitted by the female sand fly. The statistics show that according to the geographical distribution of the sand fly, around 350 million people are at risk of infection worldwide. Around 14 million people in Asia, Africa, Europe and the Americas are prone to be affected directly by this disease. It has been calculated that around 70,000 deaths a year is caused by Leishmaniasis hence putting it among the top ranking communicable diseases [One of the biological assays in the current investigational studies was against Leishmaniasis. Leishmaniasis has been reported to cause the ninth largest disease burden among infectious diseases . The disdiseases , 63.Most importantly, many of the antimicrobial compounds also manifest cytotoxicity in the eukaryotic cells, which limits their applications for therapeutic uses. This also necessitates the prior screening of test plant materials for hemolytic activities, which has also been carried out the current investigational studies with acceptable ranges.A. lasiantha were optimized using fourteen solvents systems based on their polarity to narrow down the search and isolation of bioactive compounds. Significant cytotoxic and kinases inhibiting potentials of extracts suggests bioguided isolation of anticancer compounds and further detailed studies. Antioxidant and anti-leishmanial results also suggests that the plant might be a source of phytoconstituents responsible for these activities and thus warrant further studies for isolation of compounds effective in free radicals induced degenerative disorders and Leishmaniasis.Plant materials are a robust source of potential bioactive compounds/phytochemicals with diverse physicochemical characteristics. Due to chemical diversity and natural affability of natural products, either in the form of standardized extract or as a pure compound based on ethnopharmacological properties plays a s significant role in new drug development. The most commonly employed one or two extraction solvents do not extract all the potentially bioactive constituents due to their varied polarity. The bioactive extracts of