A serpin shapes the extracellular environment to prevent influenza A virus maturation.

Interferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene SERPINE1, encoding plasminogen activator inhibitor 1 (PAI-1). By targeting extracellular airway proteases, PAI-1 inhibits IAV glycoprotein cleavage, thereby reducing infectivity of progeny viruses. This was biologically relevant for IAV restriction in vivo. Further, partial PAI-1 deficiency, attributable to a polymorphism in human SERPINE1, conferred increased susceptibility to IAV in vitro. Together, our findings reveal that manipulating the extracellular environment to inhibit the last step in a virus life cycle is an important mechanism of the antiviral response.

A nutrient-specific gut hormone arbitrates between courtship and feeding.

Animals must set behavioural priority in a context-dependent manner and switch from one behaviour to another at the appropriate moment1-3. Here we probe the molecular and neuronal mechanisms that orchestrate the transition from feeding to courtship in Drosophila melanogaster. We find that feeding is prioritized over courtship in starved males, and the consumption of protein-rich food rapidly reverses this order within a few minutes. At the molecular level, a gut-derived, nutrient-specific neuropeptide hormone-Diuretic hormone 31 (Dh31)-propels a switch from feeding to courtship. We further address the underlying kinetics with calcium imaging experiments. Amino acids from food acutely activate Dh31+ enteroendocrine cells in the gut, increasing Dh31 levels in the circulation. In addition, three-photon functional imaging of intact flies shows that optogenetic stimulation of Dh31+ enteroendocrine cells rapidly excites a subset of brain neurons that express Dh31 receptor (Dh31R). Gut-derived Dh31 excites the brain neurons through the circulatory system within a few minutes, in line with the speed of the feeding-courtship behavioural switch. At the circuit level, there are two distinct populations of Dh31R+ neurons in the brain, with one population inhibiting feeding through allatostatin-C and the other promoting courtship through corazonin. Together, our findings illustrate a mechanism by which the consumption of protein-rich food triggers the release of a gut hormone, which in turn prioritizes courtship over feeding through two parallel pathways.

Immuno-scanning electron microscopy of islet primary cilia.

The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that have a specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immunolabeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the three-dimensional ultrastructure of the cilium. Here, we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT)88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immunolabeling and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.

The coding of temperature in the Drosophila brain.

Thermosensation is an indispensable sensory modality. Here, we study temperature coding in Drosophila, and show that temperature is represented by a spatial map of activity in the brain. First, we identify TRP channels that function in the fly antenna to mediate the detection of cold stimuli. Next, we identify the hot-sensing neurons and show that hot and cold antennal receptors project onto distinct, but adjacent glomeruli in the Proximal-Antennal-Protocerebrum (PAP) forming a thermotopic map in the brain. We use two-photon imaging to reveal the functional segregation of hot and cold responses in the PAP, and show that silencing the hot- or cold-sensing neurons produces animals with distinct and discrete deficits in their behavioral responses to thermal stimuli. Together, these results demonstrate that dedicated populations of cells orchestrate behavioral responses to different temperature stimuli, and reveal a labeled-line logic for the coding of temperature information in the brain.

Presynaptic facilitation by neuropeptide signaling mediates odor-driven food search.

Internal physiological states influence behavioral decisions. We have investigated the underlying cellular and molecular mechanisms at the first olfactory synapse for starvation modulation of food-search behavior in Drosophila. We found that a local signal by short neuropeptide F (sNPF) and a global metabolic cue by insulin are integrated at specific odorant receptor neurons (ORNs) to modulate olfactory sensitivity. Results from two-photon calcium imaging show that starvation increases presynaptic activity via intraglomerular sNPF signaling. Expression of sNPF and its receptor (sNPFR1) in Or42b neurons is necessary for starvation-induced food-search behavior. Presynaptic facilitation in Or42b neurons is sufficient to mimic starvation-like behavior in fed flies. Furthermore, starvation elevates the transcription level of sNPFR1 but not that of sNPF, and insulin signaling suppresses sNPFR1 expression. Thus, starvation increases expression of sNPFR1 to change the odor map, resulting in more robust food-search behavior.

Scanning electron microscopy of human islet cilia.

Human islet primary cilia are vital glucose-regulating organelles whose structure remains uncharacterized. Scanning electron microscopy (SEM) is a useful technique for studying the surface morphology of membrane projections like cilia, but conventional sample preparation does not reveal the submembrane axonemal structure, which holds key implications for ciliary function. To overcome this challenge, we combined SEM with membrane-extraction techniques to examine primary cilia in native human islets. Our data show well-preserved cilia subdomains which demonstrate both expected and unexpected ultrastructural motifs. Morphometric features were quantified when possible, including axonemal length and diameter, microtubule conformations, and chirality. We further describe a ciliary ring, a structure that may be a specialization in human islets. Key findings are correlated with fluorescence microscopy and interpreted in the context of cilia function as a cellular sensor and communications locus in pancreatic islets.

Neuromodulation of Innate Behaviors in Drosophila.

Animals are born with a rich repertoire of robust behaviors that are critical for their survival. However, innate behaviors are also highly adaptable to an animal's internal state and external environment. Neuromodulators, including biogenic amines, neuropeptides, and hormones, are released to signal changes in animals' circumstances and serve to reconfigure neural circuits. This circuit flexibility allows animals to modify their behavioral responses according to environmental cues, metabolic demands, and physiological states. Aided by powerful genetic tools, researchers have made remarkable progress in Drosophila melanogaster to address how a myriad of contextual information influences the input-output relationship of hardwired circuits that support a complex behavioral repertoire. Here we highlight recent advances in understanding neuromodulation of Drosophila innate behaviors, with a special focus on feeding, courtship, aggression, and postmating behaviors.

Positive Regulation of Lyn Kinase by CD148 Is Required for B Cell Receptor Signaling in B1 but Not B2 B Cells.

B1 and B2 B cells differ in their ability to respond to T-cell-independent (TI) antigens. Here we report that the Src-family kinase (SFK) regulator CD148 has a unique and critical role in the initiation of B1 but not B2 cell antigen receptor signaling. CD148 loss-of-function mice were found to have defective B1 B-cell-mediated antibody responses against the T-cell-independent antigens NP-ficoll and Pneumovax 23 and had impaired selection of the B1 B cell receptor (BCR) repertoire. These deficiencies were associated with a decreased ability of B1 B cells to induce BCR signaling downstream of the SFK Lyn. Notably, Lyn appeared to be selectively regulated by CD148 and loss of this SFK resulted in opposite signaling phenotypes in B1 and B2 B cells. These findings reveal that the function and regulation of Lyn during B1 cell BCR signaling is distinct from other B cell subsets.

Low back pain and biomechanical characteristics of back muscles in firefighters.

Firefighters often experience low back pain (LBP), but their back muscle characteristics are not well studied. This study aimed to 1) compare the biomechanical characteristics of back muscles and self-reported back disabilities in frontline firefighters with and without LBP history, and 2) examine the relationships between back disability and biomechanical measurements. We recruited 42 male firefighters and assessed their perceived pain and disabilities, maximum isometric back extension strength, passive stiffness, and fatigability of the longissimus. 54.8% of the participants experienced LBP within the past year. Those indicating higher pain intensity also had greater disability as indicated by the Oswestry Disability Index. There were no significant differences in strength, stiffness or fatigability of the back muscles between firefighters with and without LBP history. Multiple linear regression analysis revealed no significant relationship between the back disability and any biomechanical or demographic measures, likely due to the high functional abilities of the participants.

Approximately 50% of the firefighters who participated in the study experienced low back pain in the past 12 months. Since the pain level was mild to moderate, most of them continued to report to work. Biomechanically, there were no differences in back muscle strength, stiffness, resistance to fatigue, or left-right symmetry between firefighters with and without back pain history. Back disabilities were not related to any biomechanical measures or demographics including age and body mass index. Overall, despite experiencing some back pain, these frontline firefighters are highly functional and did not show diminished physical or neuromuscular responses.

Primary cilia control glucose homeostasis via islet paracrine interactions.

Pancreatic islets regulate glucose homeostasis through coordinated actions of hormone-secreting cells. What underlies the function of the islet as a unit is the close approximation and communication among heterogeneous cell populations, but the structural mediators of islet cellular cross talk remain incompletely characterized. We generated mice specifically lacking ß-cell primary cilia, a cellular organelle that has been implicated in regulating insulin secretion, and found that the ß-cell cilia are required for glucose sensing, calcium influx, insulin secretion, and cross regulation of α- and δ-cells. Protein expression profiling in islets confirms perturbation in these cellular processes and reveals additional targets of cilia-dependent signaling. At the organism level, the deletion of ß-cell cilia disrupts circulating hormone levels, impairs glucose homeostasis and fuel usage, and leads to the development of diabetes. Together, these findings demonstrate that primary cilia not only orchestrate ß-cell-intrinsic activity but also mediate cross talk both within the islet and from islets to other metabolic tissues, thus providing a unique role of cilia in nutrient metabolism and insight into the pathophysiology of diabetes.

High temperature stress induced oxidative stress, gut inflammation and disordered metabolome and microbiome in tsinling lenok trout.

Tsinling lenok trout (Brachymystax lenok tsinlingensis Li) is a species of cold-water salmon that faces serious challenges due to global warming. High temperature stress has been found to damage the gut integrity of cold-water fish, impacting their growth and immunity. However, limited research exists on the causal relationship between gut microbial disturbance and metabolic dysfunction in cold-water fish induced by high temperature stress. To address this gap, we conducted a study to investigate the effects of high temperature stress (24 °C) on the gut tissue structure, antioxidant capacity, gut microorganisms, and metabolome reactions of tsinling lenok trout. Our analysis using 16 S rDNA gene sequencing revealed significant changes in the gut microbial composition and metabolic profile. Specifically, the abundance of Firmicutes and Gemmatimonadetes decreased significantly with increasing temperature, while the abundance of Bacteroidetes increased significantly. Metabolic analysis revealed a significant decrease in the abundance of glutathione, which is synthesized from glutamate and glycine, under high temperature stress. Additionally, there was a notable reduction in the levels of adenosine, inosine, xanthine, guanosine, and deoxyguanosine, which are essential for DNA/RNA synthesis. Conversely, there was a significant increase in the abundance of D-glucose 6 P. Furthermore, high temperature stress adversely affects intestinal structure and barrier function. Our findings provide valuable insights into the mechanism of high temperature stress in cold-water fish and serve as a foundation for future research aimed at mitigating the decline in production performance caused by such stress.

[Diagnosis and treatment of blunt high-grade pancreatic trauma].

The clinical data of 20 patients with blunt high-grade pancreatic trauma who were admitted to the Department of Hepatobiliary and Pancreatic Surgery of Changhai Hospital Affiliated to Naval Military Medical University from December 2003 to February 2022 were retrospectively analyzed. There were 15 males and 5 females with a median age of 39 years (range: 14-54 years). The degree of pancreatic injury was graded according to the American Association for the Surgery of Trauma (AAST) scale, including 10 cases of grade Ⅲ (50%), 8 cases of grade Ⅳ (40%), and 2 cases of grade Ⅴ (10%). Then, the strategy of diagnosis and treatment for blunt high-grade pancreatic trauma was summarized. The diagnostic rate of CT was 78.9%. Finally, 17 cases (85%) were cured and 3 cases (15%) died. Among the 10 patients with grade Ⅲ pancreatic injury, 7 cases received distal pancreatectomy and splenectomy, 1 case received distal pancreatectomy with spleen preserved, 1 case received pancreatic duct stent placement under endoscopic retrograde cholangiopancreatography (ERCP) and percutaneous catheter drainage (PCD), and 1 case received only PCD. Among 8 cases of grade Ⅳ, 3 cases underwent Roux-en-Y pancreaticojejunostomy, 1 case received distal pancreatectomy and splenectomy, 1 case underwent distal pancreatectomy with spleen preserved, 2 cases received necrotic tissue removal+external drainage of pancreatic duct+abdominal drainage, and 1 case received exploratory laparotomy and gauze packing hemostasis. For 2 cases of grade Ⅴ, 1 underwent pylorus preserving pancreaticoduodenectomy, and the other case underwent pancreaticoduodenectomy combined with right hemicolectomy and splenectomy. Therefore, the treatment of blunt high-grade pancreatic trauma should follow the individualized treatment strategy, pay attention to the control of bleeding, extensive external drainage, appropriate debridement and resection and rational application of damage control surgery, select appropriate patients for conservative treatment, and ultimately benefit the patient.

[Diagnostic value of MDM2 RNA in situ hybridization in atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma].

Objective: To investigate the value of MDM2 RNA in situ hybridization (RNA-ISH) in diagnosing atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) and dedifferentiated liposarcoma (DDL). Methods: A total of 26 ALT/WDL/DDLs diagnosed from March 2017 to May 2019 in West China Hospital, Sichuan University, Chengdu, China and 18 control cases were included. MDM2 RNA-ISH was performed on all samples and compared with the fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) regarding their performance in detecting MDM2. Results: All samples were detected successfully using the three methods. Among 26 ALT/WDL/DDLs, all cases showed MDM2 amplification and positivity for MDM2 RNA-ISH (26/26, 100%). Twenty-four (24/26, 92.3%) of the 26 tested cases were positive for MDM2 IHC while two of them were negative. Eighteen control cases were all negative for MDM2 FISH and RNA-ISH, and 15 (15/18) cases were negative for MDM2 IHC. The sensitivity and specificity of RNA-ISH were both 100%, and those of MDM2 IHC were 92.3% and 83.3%, respectively. Diffuse staining was identified in all MDM2 RNA-ISH positive ALT/WDL/DDLs, but identified in only 8/24 (33.3%) of the MDM2 IHC positive cases. Among the 11 ALT/WDL/DDL samples evaluated on tissue microarray, the positive rate of MDM2 RNA-ISH was 100% with diffuse staining in all cases. The positive rate of MDM2 IHC was 9/11 while only 1 of the 9 cases showed diffuse staining. The result of MDM2 RNA-ISH was identical to that of MDM2 FISH and was overall consistent with that of MDM2 IHC (Kappa=0.763, P<0.001). Conclusions: In ALT/WDL/DDLs, results of MDM2 RNA-ISH are highly consistent with those of FISH. MDM2 RNA-ISH is more sensitive and more specific and has more diffuse positive signals than the IHC. The findings indicate that MDM2 RNA-ISH is highly valuable for the diagnosis and differential diagnosis of ALT/WDL/DDLs.

Myogenic contraction of a somatic muscle powers rhythmic flow of hemolymph through Drosophila antennae and generates brain pulsations.

Hemolymph is driven through the antennae of Drosophila melanogaster by the rhythmic contraction of muscle 16 (m16), which runs through the brain. Contraction of m16 results in the expansion of an elastic ampulla, opening ostia and filling the ampulla. Relaxation of the ampullary membrane forces hemolymph through vessels into the antennae. We show that m16 is an auto-active rhythmic somatic muscle. The activity of m16 leads to the rapid perfusion of the antenna by hemolymph. In addition, it leads to the rhythmic agitation of the brain, which could be important for clearing the interstitial space.

[Clinical study of selection of the upper instrumented vertebra at one level caudal to upper end vertebra in patients with Lenke 5C adolescent idiopathic scoliosis].

Objective: To investigate whether the upper instrumented vertebra (UIV) can be selected at one level caudal to upper end vertebra (UEV) in Lenke type 5C adolescent idiopathic scoliosis (AIS) patients. Methods: Total of 28 Lenke 5C AIS patients who underwent selective posterior fusion in Drum Tower Hospital of Nanjing University Medical School from September 2013 to September 2015 were included. There were 4 males and 24 females, with an age of (15.0±2.0) years, the Risser sign was graded 2-5. The following imaging parameters were measured on standing full spine X-ray before, immediately after the surgery and at the last follow-up: thoracolumbar/lumbar (TL/L) Cobb angle, coronal balance, UIV translation, lower instrumented vertebra (LIV) translation, UIV tilt, LIV tilt, and thoracic apical vertebral translation (T-AVT), lumbar apical vertebral translation (L-AVT). The patients were divided into two groups: decompensation group (n=6) and non-decompensation group (n=22). Radiographic parameters and Scoliosis Research Society (SRS)-22 scores were compared between the two groups. Results: Six cases (21.4%) had proximal decompensation at the last follow-up. There were no significant differences in Risser grade(3.8±1.0 vs 3.6±1.6), baseline thoracic Cobb angle(25.8°±2.2° vs 26.3°±6.4°) and TL/L Cobb angle(43.7°±3.4° vs 45.2°±6.5°) between the two groups (all P>0.05). However, the baseline lumbar/thoracic apical vertebra translation (L-T AVT ratio) was significantly higher in patients with proximal decompensation (6.3±1.3 vs 4.0±2.0, P=0.048). Conclusion: Selecting UIV at one level caudal to UEV, would not increase the incidence of proximal decompensation in Lenke 5C AIS patients with Risser higher than grade 2, the smaller baseline L-T AVT ratio, and with thoracic compensatory curve over 15°, and can obtain satisfactory clinical results.

Effects of foot orthosis on ground reaction forces and perception during short sprints in flat-footed athletes.

Prefabricated foot orthosis (FO) is commonly worn for flat foot management. This study aimed to investigate the kinetic and perceptual effects of wearing prefabricated FO among flat-footed athletes during bouts of sprints. Twenty male sprint-based sports athletes who had flat foot bilaterally ran at three test speeds (5, 6, 7 m/s) under two conditions: prefabricated FO and sham FO. Ground reaction force (GRF) variables and subjective perceptions were recorded. Kinetic variability of GRF variables were computed to indicate step-to-step variance. Biomechanically, wearing prefabricated FOs increased vertical impact force (p =.005), loading rate (p =.001), and kinetic variability of peak propulsive force (p =.038) and loading rate (p =.019) during sprinting speeds across 5 to 7 m/s. Subjectively, prefabricated FO provided better arch support (p =.001) but resulted in reduced forefoot cushioning (p =.001), heel cushioning (p =.002), and overall comfort (p =.008).

Receptor-like tyrosine phosphatases CD45 and CD148 have distinct functions in chemoattractant-mediated neutrophil migration and response to S. aureus.

Neutrophils, critical innate immune effectors, use bacterial-derived chemoattractant-induced G protein-coupled receptor (GPCR) signaling for their pursuit of bacteria. Tyrosine phosphorylation pathways and receptor-like tyrosine phosphatases (RPTPs) are rarely considered in chemoattractant-mediated GPCR signaling. Here, we report that two RPTPs, CD45 and CD148, previously shown to share redundant roles in positively regulating Src family kinases (SFKs) in immunoreceptor signaling pathways in B cells and macrophages, are critical in the neutrophil response to S. aureus infection and, surprisingly, in chemoattractant-mediated chemotaxis. Remarkably, deficiency in either of these RPTPs influenced neutrophil GPCR responses in unique ways. Our results reveal that CD45 positively while CD148 positively and negatively regulate GPCR function and proximal signals including Ca(2+), phosphatidylinositol 3'OH kinase (PI3K), and phospho-extracellular regulated kinase (pERK) activity. Moreover, our results suggest that CD45 and CD148 preferentially target different SFK members (Hck and Fgr versus Lyn, respectively) to positively and negatively regulate GPCR pathways.

Between-limb asymmetry in kinetic and temporal characteristics during bilateral plyometric drop jumps from different heights.

This study investigated the between-limb asymmetry in kinetic and temporal characteristics during bilateral plyometric drop jumps from different heights. Seventeen male basketball players performed drop jumps from 3 heights on two platforms in randomized orders. Vertical ground reaction force data were analysed with respect to the lead limb (i.e. the limb stepping off the raised platform first) and trail limb. Peak forces and loading rates of each limb were calculated. The absolute time differential between the two limbs at initial ground contact and takeoff were determined. The frequency of symmetrical landing and taking off with "both limbs together" were counted using 3 time windows. Results showed that the lead limb displayed higher peak forces and loading rates than the trail limb across all heights (p <.05). As drop height increased, the absolute time differentials decreased at initial ground contact (p <.001) but increased at takeoff (p =.035). The greater the preset time window, the more landings and takeoffs were classified as bilaterally symmetrical. In conclusion, higher drop heights allowed subjects to become more bilaterally symmetrical in the timing of landing but this reduction in temporal asymmetry did not accompany with any reduction in kinetic asymmetry.

[Alveolar crest and relevant analysis of labial side of anterior teeth on skeletal Angle class III patients].

OBJECTIVE: To measure the distance from cemento-enamel junction (CEJ) to alveolar crest (AC) of labial side of anterior teeth on skeletal Angle class III patients under direct vision during periodontal bone augmentation surgery and to make relevant analysis to find the relevant factors. METHODS: In the study, 46 skeletal Angle class III patients (10 males and 36 females) received periodontal bone augmentation surgery of anterior teeth were included, with 67 jaws (27 maxillae and 40 mandibles) and 400 anterior teeth (161 maxillary anterior teeth and 239 mandibular anterior teeth). The mean age was 23.65 years. Maxillary anterior teeth consisted of 54 central incisors, 53 lateral incisors and 54 canines. Mandibular anterior teeth consisted of 79 central incisors, 80 lateral incisors and 80 canines. CEJ-AC was measured in three sites (mesial sites, central sites and distal sites) by Williams periodontal probes during periodontal bone augmentation surgery under direct vision by the same researcher. RESULTS: The average CEJ-AC of 400 anterior teeth was (2.21±1.48) mm. The average CEJ-AC of maxillary anterior teeth was (1.72±1.13) mm, more than (2.54±1.60) mm of mandibular anterior teeth (P<0.05). The average CEJ-AC of canines was (2.42±1.78) mm, more than (2.06±1.27) mm of central incisors or (2.16±1.32) mm of lateral incisors (P<0.05). The average CEJ-AC of central sites was (3.04±2.01) mm, more than (1.79±0.86) mm of mesial sites or (1.81±0.89) mm of distal sites (P<0.05). CEJ-AC of 233 anterior teeth was more than 2 mm, accounting for 58.25%, and 117 anterior teeth with dehiscence were found, accounting for 29.25%. Multilevel and multivariate Logistic regression showed age, jaw, tooth and site were the relevant factors to the position of alveolar crest. CONCLUSION: The position of alveolar crest of skeletal Angle class III patients who received periodontal bone augmentation surgery was lower than that of the general population, causing periodontal risks during decompensation orthodontics therapy before orthognathic surgery. The position of alveolar crest was lower in older patients than in younger patients, in mandibular teeth than in maxillary teeth, in canines than in central incisors or lateral incisors, and in central sites than in mesial sites or distal sites of labial side, which showed much higher risk.

Effect of caffeine ingestion on free-throw performance in college basketball players.

BACKGROUND: It is currently unclear whether pre-exercise caffeine ingestion can improve free-throw shooting performance, a vital skill in basketball. The purpose of this study was to investigate the effects of caffeine on free-throw shooting performance in college-aged basketball players. METHODS: Twelve males (23.1 ± 1.9 years; 180.1 ± 8.8 cm; 77.1 ± 12.4 kg) and six females (22.0 ± 1.3 years; 169.4 ± 8.9 cm; 67.0 ± 11.1 kg) who competed at the college level ingested 6 mg per kg of body mass of (a) caffeine or (b) maltodextrin (placebo) on two separate occasions in a random order. After 60 min, they performed five sets of a match-simulated basketball protocol comprising six sideline-to-sideline sprints on a standard basketball court followed by two free-throws after each set. The number of successful shots was counted. Heart rate and rating of perceived exertion (RPE) after each sprint set were also recorded. RESULTS: Caffeine ingestion did not improve overall free-throw success (caffeine = 6.1 ± 1.7 vs. placebo = 5.5 ± 2.0; p = 0.34) compared with placebo across all five sets. There was no change in shooting accuracy across sprint sets in either trial despite significant increases in both heart rate and RPE. Caffeine increased heart rate (p = 0.02) but had no effect on RPE (p = 0.57) across five sets compared with placebo. CONCLUSIONS: Ingestion of 6 mg of caffeine per kg of body mass did not improve basketball free-throw performance. Free-throw performance did not deteriorate with increasing number of sprint sets.