Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) describes an indolent, under-recognised and poorly understood pulmonary condition with fewer than 200 reported cases across the literature. Currently, cases are diagnosed following a biopsy of the primary lesion, with treatment options centring on symptomatic benefit as opposed to targeting the underlying aetiology. Classically, DIPNECH lesions have been described as slow growing and benign, but with growing awareness of the condition, reports of metastatic disease with significant symptomatic burden have been reported. However, effectively addressing the subset of DIPNECH patients with greater metastatic potential remains an unmet clinical need.Due to the similarities between DIPNECH and carcinoid patients, several centres have considered using somatostatin analogues to not only help symptomatically but also to initiate tumour regression. However, to date, there are limited biomarkers to help evaluate the benefit of such options. In this review, we consider the use of pulmonary function tests (PFTs) to help quantify the benefit of somatostatin analogues. Although much of the evidence stems from small single-centre studies, the use of PFTs within the treatment pathway for both localised and metastatic DIPNECH represents a meaningful improvement from subjective monitoring of disease. Characteristics of diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) DIPNECH describes an indolent, exceptionally rare, and inadequately understood pulmonary disorder with under 200 cases across the literature . The conHistologically, DIPNECH is recognised in a range of forms following a biopsy. It is accepted as a ‘generalised proliferation of scattered neuroendocrine cells, small nodules (neuroendocrine bodies) or a linear proliferation of pulmonary neuroendocrine cells’ . The preIn contrast to extrapulmonary carcinoid tumours where no obvious sex preference has been identified, DIPNECH occurs predominantly in females 9:1) and at a higher mean age of 66 years  and at a,6. CliniWhilst DIPNECH remains a solely histological diagnosis, characteristic high-resolution computed tomography (HRCT) findings can aid identification. Radiologically lesions are almost always bilateral where suggestive features of airway disease including bronchial wall thickening and bronchiectasis are commonly observed. Mosaic perfusion is considered the most significant and suggestive feature of DIPNECH with a better appreciation of expiratory CT scans . AdditioCurrent treatment optionsCurrent management options are limited and require a multi-disciplinary approach coupled with life-long surveillance. Surgical resections in patients with dominant nodules offer symptomatic relief, whilst also reducing the progression toward severe disease and metastatic spread. Two case reports in 2011 and 2019 both described cases of parenchymal sparing wedge resections in patients with DIPNECH ,10. HoweThe lack of licenced, targeted pharmacological therapies for DIPNECH leaves clinicians with treatment options aimed at alleviating symptoms rather than disrupting the underlying aetiology. For instance, patients are given corticosteroids and a combination of short and long-acting beta-agonists to relieve dyspnoea and improve obstructive lung function but evidence on efficacy within this population remains scarce ,15,16. RMutations and dysregulation of the mechanistic target of the rapamycin (mTOR) pathway have been well described in conventional neuroendocrine tumours (NETs) . The pre 111In-octreotide scintigraphy (an OctreoScan using radiolabeled with indium-111) within the lungs of a DIPNECH patient [Similarly, the expression of somatostatin receptors (SSTR), particularly SSTR2 is well documented in pulmonary carcinoids advocating the use of somatostatin analogues (SSAs) as a targeted therapy ,22. One  patient . Possibl patient ,25. This patient .Studies monitoring the effects of SSAs on DIPNECH have described largely positive findings. One retrospective study of five DIPNECH patients started on octreotide identified drastic improvements in cough from four patients . SimilarThe increased recognition and understanding of DIPNECH offers an opportunity for innovative therapeutics to continue to be trialled as a means of improving patient prognoses. However, the true efficacy of these therapeutics is often negated due to the subjectivity involved in measuring clinical response. The use of patient-reported outcomes or validated quality-of-life questionnaires have also been advocated, which would provide more objective and replicable metrics . CorroboPFTs as a BiomarkerPFTs are a non-invasive set of investigations that quantify lung function. PFTs recognise obstructive and restrictive patterns of lung disease allowing accurate diagnoses and prompting suitable subsequent management. Spirometry is a well-established PFT providing three measurements: volume, time and flow. It is objective, sensitive to early change and reproducible, thus making it an appropriate biomarker for this case study .Spirometry Produces the Following MeasurementsForced expiratory volume in 1 second (FEV1) is the total volume of air breathed out in a second, measured in litres. Forced vital capacity (FVC) is the total volume of air breathed out in one continuous breath, measured in litres. Vital Capacity (VC): The total volume of air breathed whilst at rest, measured in litres. FEV1/FVC ratio is the FEV1 divided by the FVC used in identifying obstructive, restrictive and mixed patterns of lung function. The values derived from spirometry are compared to pre-existing data, either reference values or predicted values to allow interpretation. Reference values are formulated from population data consisting of multiple influential variables such as age, ethnicity, smoking status, height, weight, sex, etc. This data is then compiled into equations that generate reference values . Within Testing the Validity of SpirometryWithin a given test population there is known heterogeneity amongst exhalations during FVC manoeuvres. A 1982 study best represented the diversity within FVC manoeuvres using 24 standard volume time waveforms . These wThe Discovery of SpirometryThe word spirometer originates from the Latin constituents of spiro (to breathe) and meter (to measure). Evidence of a physical spirometer dates back to a device invented by the British surgeon John Hutchinson in the 1840s. The large device used a calibrated bucket submerged in water which was attached to a breathing tube. Exhaled air in the tube could then be accurately measured . HutchinAlmost a century later, in 1933, German doctor J. Hermannsen first recorded the maximum exhilaration during continuous voluntary effort ,35.Over time, the need to establish homogonous nomenclature led to ‘volume expiratoire maximum  seconde’ (VEMS) replacing CPUE . FinallyAn Overview of the Current Uses of SpirometrySpirometry is used extensively in respiratory medicine. It can be used to aid diagnosis, initiate management, and monitor conditions during follow-up. Primarily, the FEV1/FVC ratio defines the pattern of lung disease. A ratio >0.70 is associated with restricted lung disease, whereas a ratio <0.70 is associated with obstructive lung disease. Spirometry in COPDA post-bronchodilator FEV1/FVC ratio <0.70 is classically considered diagnostic for COPD, a condition characterised by irreversible and progressive airflow resistance . Once anA recent literature review analysed the correlation between PFTs and patient-reported outcomes (PROs). These PROs were derived from the Transition Dyspnoea Index (TDI); an indicator of change in dyspnoea from baseline and The St. George’s Respiratory Symptom Questionnaire (SGRQ), which captures information on health-related quality of life and the impact of disease on well-being and activities of daily living. Their results found that patients with greater improvements in trough FEV1 after treatment had better SGRQ and TDI scores, fewer COPD exacerbations and also used less rescue pack medications . TherefoConversely, numerous studies have highlighted the association between poorer lung function and increased mortality. For example, a study following >1,100 patients over a 29-year period found that predicted FEV1 of normal (FEV1%pred) adjusted to baseline characteristics  was inversely related to all-cause mortality across both males and females . AnotherSpirometry in AsthmaAsthma is a chronic respiratory condition with characteristic airway inflammation and hyperresponsivity. Clinically, it presents as a symptom complex of dyspnoea, auditory wheeze, chest tightness and cough with objective evidence of reversible airway obstruction . SpiromeSpirometry and PFTs in DIPNECHDIPNECH is predominantly characterised by an obstructive pattern of lung disease . AlthougSupporting this hypothesis, a study measuring the change in PFTs following radiotherapy in non-small-cell lung cancer (NSCLC) patients showed significant improvements in FEV1 after radiotherapy-induced tumour regression . SimilarConsidering the dispersed nature of DIPNECH nodules found on presentation, targeted radiotherapy may be limited to the largest nodules and therefore offers a partial treatment option. Consequently, alternative therapeutics that can induce homogonous tumour regression should be considered. The antiproliferative properties of SSAs have been demonstrated in large clinical trials and thus offer a viable treatment option for DIPNECH . Hence, One study initiated 42 DIPNECH patients with SSAs . At baseNotable issuesThe number of DIPNECH studies using PFTs as a biomarker for measuring response to treatment remains low. Amongst these few studies, several noteworthy issues should be raised. For instance, in two studies, there was a significant decrease in the number of patients for whom follow-up PFT data were available ,51. ThisFurthermore, multiple studies have reported data on post-treatment FEV1 without specifying the exact elapsed time after initiating treatment till a repeat FEV1 was measured. Moreover, the frequency of FEV1/PFT testing during follow-up is rarely specified and likely to be highly variable among the studies ,23,24,52The use of PFTs to objectively measure treatment response represents a meaningful improvement from previous studies which primarily reported subjective findings . HoweverTreatment success defined by an improvement in PFT following treatment is influenced by numerous factors. These include patient characteristics, the efficacy of treatment and the heterogeneity of DIPNECH between individuals. However, for treatment-related changes in PFT to manifest, a sufficient follow-up duration is needed. A 2020 study of 44 patients reported no significant improvements in PFT for half their SSA sample but the median follow-up time was only 30 months . Short fSurgical resection plays a common role in the initial management of suspected DIPNECH patients with dominant nodule(s). Resection aims to not only remove the dominant nodule(s) and thus reduce the progression of the disease, but also allows for histopathological analysis needed for a definitive diagnosis. In one study, nine patients underwent thoracotomy and resection of the dominant nodule prior to any initiation of pharmacological treatment . The 202Furthermore, treatment often comprised concomitant medications in addition to the SSAs. These included steroids, inhalers and narcotic cough medicines aimed at alleviating symptoms . Their uIn this review, we have described how PFTs, and in particular spirometry, can be adopted from other areas of respiratory disease management and fundamentally incorporated into DIPNECH management. PFTs help evaluate treatment efficacy and are used to effectively monitor disease progression. Currently, SSA remains licenced for palliation of symptoms but the growing number of reports utilising these drugs as an initial treatment for DIPNECH coupled with evidence for their antiproliferative abilities influenced our choice of therapeutic. Evidence that SSTR expression increases with tumour differentiation corroborate with studies showing uptake accumulating within dominant nodules during SSTR imaging. Therefore, we can hypothesise that SSAs may have a greater effect on more advanced cases of DIPNECH where greater differentiation across the spectrum of pre-cancerous to cancerous cells are present or in cases where surgical resection of the dominant nodule was not possible. Nevertheless, incorporating PFTs as standard management protocol helps objectively monitor patients and thus allows clinicians to gather more data about the pathophysiology of DIPNECH and the efficacy of treatments.Many studies are limited by sample size, owing to the rarity of the disease and the result of common misdiagnoses of patients. However, the clear acceleration in recognition of DIPNECH in recent years is hugely promising and will likely yield future targeted therapies that have the potential to change the current outlook. Whether SSAs will be included in these future therapies remains to be seen. In any case, the incorporation of PFTs into DIPNECH management represents a huge step forward allowing clinicians to accurately stratify patients and providing the opportunity for more personalised therapies.


Decreased loading of the paretic lower limb and impaired weight transfer between limbs negatively impact balance control and forward progression during gait in individuals post-stroke. However, the biomechanical and neuromuscular control mechanisms underlying such impaired limb loading remain unclear, partly due to their tendency of avoiding bearing weight on the paretic limb during voluntary movement. Thus, an approach that forces individuals to more fully and rapidly load the paretic limb has been developed. The primary purpose of this study was to compare the neuromechanical responses at the ankle and knee during externally induced limb loading in people with chronic stroke versus able-bodied controls, and determine whether energy absorption capacity, measured during induced limb loading of the paretic limb, was associated with walking characteristics in individuals post-stroke. Results revealed reduced rate of energy absorption and dorsiflexion velocity at the ankle joint during induced limb loading in both the paretic and non-paretic side in individuals post-stroke compared to healthy controls. The co-contraction index was higher in the paretic ankle and knee joints compared to the non-paretic side. In addition, the rate of energy absorption at the paretic ankle joint during the induced limb loading was positively correlated with maximum walking speed and negatively correlated with double limb support duration. These findings demonstrated that deficits in ankle dorsiflexion velocity may limit the mechanical energy absorption capacity of the joint and thereby affect the lower limb loading process during gait following stroke. Approximately one in three stroke survivors experience long-term disability, which makes stroke one of the leading causes of disability in the United States. Among people with hemiparesis due to stroke, reduction in magnitude, duration, and rate of loading of the paretic lower limb during functional activities are commonly observed and persists after the injury. For example, individuals with chronic stroke commonly bear less weight on the paretic leg than the non-paretic leg during quiet standing, sit-to-stand, and walking4, have shorter paretic single stance time than non-paretic during walking5, and take longer duration to transfer body weight from the non-paretic limb to the paretic limb during walking4. Deficits in paretic lower limb loading are associated with higher postural sway, lower overall motor function, and an increased incidence of falling10. Improvements in loading capacity of the paretic lower limb are also associated with increased balance control, walking speed, and reduced risk of falls13. In addition, the ability to transfer body weight between limbs is an important biomechanical factor that relates to limb loading14. Longer duration and decreased amplitude of weight transfer from the non-paretic leg to the paretic leg have been reported in individuals post-stroke that negatively impacts forward progression15. Thus, improving weight transfer and paretic lower limb loading ability are major focuses in post-stroke rehabilitation.Over 795,000 people are affected by stroke every year in the United States, which resulted in nearly $53 billion in overall costs between 2017 and 201817. Another study used a shoe insole installed beneath the non-paretic leg to shift the body center of mass toward the paretic side during daily activity18. After 6 weeks of interventions focused on weight shifting, significant improvements in lower limb loading symmetry along with increased walking speed were found19. Overall, these findings suggest that individuals post-stroke have the capacity to improve paretic lower limb loading function. However, despite the moderate improvement achieved with these interventions, previous results generally failed to show long-term success of the recovery20. This is partly due to the limited understanding of biomechanical and neuromuscular control mechanisms underlying impaired paretic limb loading ability.Different approaches have been attempted to restore paretic limb loading and weight transfer ability post-stroke. For example, de Haart and colleagues reported that during standing, loading asymmetry between the lower limbs was reduced after 12 weeks of conventional physical therapy23. In particular, the ankle and knee joints are the primary impact energy absorbers during rapid limb loading25 and therefore contribute to a smooth weight transfer process22. During the double support phase of gait, body weight is normally transferred from the trailing limb to the leading limb, requiring a coordinated push-off work from the trailing limb and corresponding energy absorption at the leading limb to ensure an efficient step-to-step transition27. Thus, deficits in energy absorption ability may disrupt the weight transfer process and ultimately impact walking speed.During movements such as walking, landing, and protective stepping, functional limb loading requires the lower extremity to regulate the impact force and absorb or dissipate the kinetic energy28. Other studies have reported increased joint stiffness and co-contraction of the agonist and antagonist muscles at the ankle and knee joints during voluntary movements in individuals post-stroke compared to neurologically intact adults33. These observations suggested potential neuromechanical factors underlying impaired limb loading and weight transfer following stroke. However, whether and how these neuromechanical factors affect energy absorption during rapid limb loading following stroke remains unclear. Furthermore, the relationships between energy absorption ability during perturbation-induced limb loading and functional gait characteristics remain to be determined.Using a novel perturbation to impose rapid loading towards the paretic lower extremity, we have previously identified stroke-associated abnormalities in ankle and knee joint kinematicsAccordingly, the primary purpose of this study was to compare the rate of energy absorption and agonist–antagonist muscle co-contraction at the ankle and knee joints during a perturbation-induced limb loading in individuals post-stroke versus age-matched controls. In addition, we aimed to determine whether the rate of energy absorption measured during induced limb loading assessment was associated with walking characteristics in individuals post-stroke. We hypothesized that: (1) Compared to healthy age-matched controls, reduced rate of energy absorption and increased co-contraction of the agonist and antagonist muscles at ankle and knee joints would be observed in people with chronic stroke; and that (2) the rate of energy absorption (i.e. power) at ankle and knee joints during induced limb loading would negatively correlate with double support duration during walking and positively correlate with gait speed in people with chronic stroke.Fifteen individuals post-stroke  along with fifteen age-matched healthy controls  participated in the study. The inclusion criteria of individuals post-stroke were (1) more than 6 months since last stroke, (2) ability to walk 10 m with or without a walking aid, and (3) ability to stand without any support for 5 min. Able-bodied controls were included if they had no self-reported history of any neurological injuries or any current musculoskeletal conditions. Exclusion criteria for both groups were: (1) underlying medical history precluding participation in regular exercises, (2) unable to follow instructions, and (3) self-reported pregnancy. The study was approved by the University of Maryland Baltimore Institutional Review Board and all participants provided written informed consent to participate. All experiments were performed in accordance with relevant guidelines and regulations.In the final analysis, data for one participant with stroke were excluded due to technical issues in EMG recording. In addition, because we were primarily focusing on the biological factors that affect limb loading, data for two additional participants with chronic stroke who wore an ankle–foot orthosis (AFO) that may affect ankle joint movement were excluded .34. From our preliminary result, the drop distance of 4.3 cm was capable of inducing a peak loading of approximately 90% body weight on the dropped lower limb without causing safety concerns.Next, an imposed limb loading assessment was performed by using two adjacent perturbation platforms (height ~ 37 cm) each placed above a force platform . Each of the perturbation platforms included a detachable standing support surface held securely on top of a support frame using ten electromagnets . Disengagement of the magnets via a computer-controlled external circuit released the detachable surface causing it to drop 4.3 cm vertically. A layer of noise-reduction material (thickness ~ 5 mm) was glued at the bottom of the detachable surface to minimize a sound induced startle response, which is commonly triggered by a sudden acoustic stimulation over 70 dBParticipants stood with one foot on each perturbation platform and were instructed to stand naturally with their weight evenly distributed on each leg. The vertical ground reaction forces were monitored to ensure symmetrical limb loading prior to perturbation onset. During each trial, one of the detachable surfaces was released at an unpredictable time, causing a sudden body weight transfer towards the dropped side and induced loading of the perturbed lower limb Fig. . Partici35 were applied with additional markers attached bilaterally on the 5th metatarsal, medial elbow, greater trochanter, medial knee, medial malleoli, and the detachable surfaces of the perturbation platform. Marker data were sampled at 150 Hz for 7 s. Two force platforms  each located beneath a perturbation platform were used to record ground reaction forces (GRF) and center-of-pressure (COP) data. Force data were sampled at 1500 Hz for 7 s. Muscle activation patterns were recorded bilaterally with surface electrodes  placed over the gluteus medius (GM), vastus lateralis (VL), biceps femoris (BF), medial gastrocnemius (MG), and tibialis anterior (TA) muscles. EMG signals were sampled at 1500 Hz for 7 s.Kinematic, kinetic, and electromyographic signals were only recorded during the imposed limb loading assessment and not during gait assessment. A 10-camera Vicon motion capture system  was used to capture the 3-dimensional body segment position. Plug-in Gait marker setOverground walking speed, double support duration (% gait cycle) when the paretic limb was the leading limb, and double support duration (% gait cycle) when the non-paretic limb was the leading limb were calculated by the GAITRite software during both self-selected and maximum speed conditions.Kinematic and kinetic data were low pass filtered at a cutoff frequency of 6 and 30 Hz, respectively. EMG signals were bandpass-filtered at 16–500 Hz, de-meaned, full-wave rectified, and lowpass-filtered at 20 Hz. All the data were processed offline with a fourth-order Butterworth filter.36  was used to calculate body center of mass (CoM) position, sagittal plane ankle and knee joint angle, torque and power using Visual3D software . Downward kinetic energy of the CoM and ankle and knee joint angular velocity were then calculated. The ground reaction forces, joint torque and power, and kinetic energy were all normalized to body mass. Because we were interested in the interval when the downward kinetic energy was absorbed, the impact absorption phase was defined as the time period from the first maximum downward kinetic energy of CoM to the first minimum downward kinetic energy of CoM following a perturbation36 Fig. . Peak va37. Muscle co-contraction was calculated using Eq.  and the impact absorption phase  and knee joint (VL and BF) muscles and was normalized to the duration of each phase.Co-contraction was calculated between an agonist–antagonist pair, and “lower EMGase Fig. . Next, t41 to control the false discovery rate at 5% for multiple comparisons. Tukey’s HSD approach was performed to compare the estimated marginal means between each pair of groups. The significance level was set at an α of 0.05. Estimated marginal means are reported as ‘mean’ in the results. The percent difference between individuals post-stroke and the controls was calculated as the ratio of the difference of estimated marginal means between groups over the estimated marginal mean value of the control group.A linear mixed-effect model using lower extremity  as a fixed factor nested under a random factor of subject was used to test the differences of (1) peak ankle and knee joint rate of energy absorption, angular velocity, and torque during the impact absorption phase, and (2) ankle and knee joint muscle co-contraction index during the baseline and the impact absorption phase between groups. The p-values were adjusted using the Benjamini–Hochberg procedure42.If differences in the rate of energy absorption at the ankle or knee joint were detected, stepwise linear regression was performed to determine the relative contributions of the angular velocity and torque to the rate of energy absorption at the joint for each group. Simple linear regression was used to investigate the association between peak ankle rate of energy absorption during induced limb loading assessment and (1) self-selected and maximum walking speed, and (2) double support duration when the corresponding limb was the leading limb at self-selected and maximum speeds in individuals post-stroke. All the statistical analyses were conducted in RReduced peak rate of energy absorption at the ankle joint was shown in both the paretic and non-paretic limb compared to controls  contributed more to rate of energy absorption than joint torque  for the control group. In contrast, ankle joint torque was the primary contributor to rate of energy absorption for both the paretic and non-paretic limb .No differences in co-contraction index were detected between individuals post-stroke and controls in ankle and knee joints. Higher co-contraction index during the impact absorption phase was found at the ankle and knee joints in the paretic side compared to the non-paretic side in individuals post-stroke  and double support duration  when walking at maximum walking speed in participants with chronic stroke. No correlation was found between peak paretic ankle rate of energy absorption and speed or double support duration when walking at a self-selected speed. No significant correlation was found between peak ankle rate of energy absorption at the non-paretic limb and speed or double support duration when walking with self-selected or maximum walking speed (Fig. Peak paretic ankle rate of energy absorption during induced limb loading was correlated with walking speed (R28 by demonstrating that deficits in ankle dorsiflexion velocity during limb loading may limit the maximum mechanical energy absorption capacity of the joint and thereby affect weight transfer process and maximum walking performance following stroke.In the present study, we assessed the biomechanical and neuromuscular factors that affect impact energy absorption during perturbation-induced limb loading in individuals post-stroke versus age-matched healthy controls. Our results revealed a reduced peak rate of energy absorption in the ankle joint in both paretic and non-paretic limbs among individuals post-stroke compared to controls, likely due to the limited dorsiflexion velocity at the ankle joint during the induced limb loading. In addition, individuals post-stroke who had higher rate of energy absorption at the paretic ankle joint during induced limb loading assessment also had higher speed and shorter double support duration when walking at maximum speed. These findings extend previous work43. During gait, the energy absorbed by the leading limb is dependent on the push-off capacity of the contralateral trailing limb44. Because a higher rate of energy produced by the non-paretic trailing limb was reported compared to unimpaired controls in the study by Farris et al.43, the observed increases in the paretic ankle rate of energy absorption might have reflected the greater push-off power from the non-paretic trailing limb. In the present study, limb loading perturbations were delivered separately to each lower extremity, which allowed us to examine the rapid loading response of each limb in a more independent manner. With this approach, the impaired impact absorption ability in both paretic and non-paretic ankle joints was identified.Our findings showed that when undergoing rapid loading, both paretic and non-paretic ankle joints had a reduced rate of energy absorption compared to controls. These results were in contrast to a previous gait study that reported a higher rate of energy absorbed by the leading paretic limb ankle compared to healthy controls during step-to-step transition43, our result suggested that the ankle joint plays an important role in energy absorption during weight transfer. In addition, increased double support duration and decreased walking speeds are commonly observed following stroke45. Our result showed that participants with a higher energy absorption capacity at the paretic ankle joint during induced limb loading also had a shorter double support phase with the paretic limb as the leading limb during maximal walking speed, suggesting that greater paretic ankle energy absorption capacity could potentially contributed to more efficient weight transfer during locomotion. Moreover, participants with greater paretic ankle rate of energy absorption capacity also demonstrated greater maximum walking speed. These findings suggest that a deficit in the rate of energy absorption at the ankle joint may be an important target for rehabilitation that aims to improve limb loading and weight transfer efficiency post-stroke, especially for those stroke survivors with slower maximum walking speed. However, it is worth noting that the causation between paretic ankle rate of energy absorption and walking performance in individuals post-stroke remains to be determined. Future studies will investigate whether improving energy absorption capacity could lead to improvement in walking performance.Consistent with a previous study that reported more energy absorbed at the paretic ankle joint than the knee joint during step-to-step transition in gait after stroke46. When walking with self-selected speed, the demand for energy absorption is submaximal during weight transfer, whereas walking at maximum speed likely demands greater energy absorption capacity of the paretic ankle. Given that the loading rate during induced limb loading assessment was much larger than that during walking under self-selected speed, the biomechanical characteristic identified during induced limb loading may be more reflective of the participants’ maximum capacity rather than preferred movement speed used in gait. Previous studies aiming to identify mechanisms that associate with maximum walking speed post-stroke have primarily focused on the power and force generation capacity at the paretic limb48. Our results expand upon these previous studies by demonstrating that, in addition to decreased force generation, reduced energy absorption ability could be a limiting factor for walking speed modulation capacity post-stroke.We did not find a significant relationship between ankle rate of energy absorption during induced limb loading assessment and self-selected walking speed in people post-stroke. This could be due to the different limb loading rate between tasks. During the induced limb loading, the assessed limb was loaded with an average of 98% bodyweight within 230 ms, which was about 20% shorter than the time to reach similar limb loading during walking at an average speed of 0.88 m/s in people post-stroke49, which has been associated with hyperreflexia50, increased stiffness of the joint and muscle mechanical properties52, and increased muscle co-contraction53. Although the cause of reduced ankle dorsiflexion velocity in individuals post-stroke shown during the induced limb loading task could be the combination of all these factors, the weight of each factor could be different between the paretic and the non-paretic limb. We found that the co-contraction index was higher in the paretic ankle compared to the non-paretic side, indicating a higher contribution of the neuromuscular factor to the reduced dorsiflexion velocity at the paretic ankle. It is important to note that we also found a higher baseline co-contraction index in the paretic ankle compared to the non-paretic side. Thus, our results may reflect that abnormal baseline muscle activity could carry over to the impact absorption phase. Participants with stroke used ankle joint torque production as the primary strategy to absorb energy, rather than using ankle angular velocity as seen in able-bodied individuals. This altered strategy could indicate that individuals post-stroke utilized their retained ankle plantarflexion torque to compensate for deficits in ankle joint movement velocity to regulate impact absorption. Interventions that incorporate rapid ankle movements to enhance rate of energy absorption are likely important for restoring limb loading ability for individuals post-stroke. Importantly, reduced rate of energy absorption and dorsiflexion velocity at the ankle joint were observed in both the paretic and the non-paretic limbs compared to healthy controls. Thus, motor recovery evaluation and limb loading training for both limbs should be considered in the post-stroke rehabilitation program.Joint torque and angular velocity are two elements that contribute to the rate of energy absorption. We found that reduced dorsiflexion velocity at both the paretic and non-paretic ankle joint appeared to be the primary factor that limits rapid limb loading following stroke. The decreased joint velocity could be related to the exaggerated resistance to passive movement post-stroke56. Results from the present study showed that ankle plantarflexion torque appeared to be preserved in participants with stroke during impact absorption. This discrepancy could be due to different approaches used across studies. In this study, the ankle plantarflexor muscles performed an eccentric contraction with a high velocity during rapid limb loading. Compared to a concentric contraction, joint torque production was less impaired during eccentric motion in individuals post-stroke, especially at higher speeds56. In addition to the contraction type, the joint position could also contribute to the preserved ankle plantarflexion torque in people post-stroke during induced limb loading. Previous studies have shown that the higher reflex-mediated and intrinsic muscle stiffness in the paretic ankle plantar flexors is most prominent when assessed in a dorsiflexed position57. During induced limb loading assessment, the ankle joint was dorsiflexed during the impact absorption phase, which could lead to a relatively higher plantarflexion torque compared to other studies.Previous studies have reported that muscle strength during isometric and concentric contraction is significantly reduced after stroke58. Our results support these previous findings by showing that abnormalities in the ankle joints are likely primary limiting factors of vertical limb loading ability following stroke. It is worth noting that in gait studies, the knee joint was reported to be the main impact absorber during normal walking59. A major difference between the present study and gait studies is the body configuration during limb loading. In the present study, participants were instructed to stand with a natural parallel stance. During post analysis, we observed that the ground reaction force vectors were oriented closely to the knee joint center of the loaded limb and therefore had a relatively small external moment arm and external torque applied to the knee joint. During dynamic gait, the leading limb is normally positioned in front of the body center of mass during initial contact and the ground reaction force vector is further away from the knee joint center. Thus, greater external knee torque was observed during walking compared to the present study60. These results warrant future studies to investigate the response to limb loading perturbation in different postures during gait. In particular, imposing limb loading to the leading limb during the double support phase of gait could induce ground reaction forces in the anterior–posterior direction and likely requires increased knee joint torque production and energy absorption.Although we found a higher agonist–antagonist co-contraction at the paretic knee joint compared to non-paretic side, we did not identify any difference in knee joint kinetics and kinematics between groups. This is likely in part due to the task performed in this study. During vertical limb loading movements, the ankle has been reported to be the primary shock absorbing jointThere are limitations in this study. First, the perturbation was a relatively small vertical displacement of standing support surface to ensure participants’ safety. Although the perturbation intensity was sufficient to induce loading with an average of 98% bodyweight to the perturbed limb and identify neuromechanical abnormalities during limb loading, our results may not be generalizable to tasks with different intensities. Thus, further testing of a broader range of intensities may generate additional useful information. Secondly, only spatiotemporal gait characteristics were captured in this study. Although we found that deficits in ankle energy absorption capacity during induced limb loading were associated with longer double support duration during gait, the direct relationship between abnormalities in ankle and knee joint mechanics during gait versus induced limb loading remains to be determined. In addition, the reactive limb loading response tested in the present study could be inherently different from proactive limb loading during gait. To better understand whether our findings could be directly applicable to gait, future studies investigating forced limb loading during continuous gait is warranted. Last but not least, we did not include participants who wore an AFO during the imposed limb loading task in the final result. In the post analysis, we found that including participants with AFO produced results that were generally consistent with the current result, with reduced p-values in between-group comparisons of ankle power and angular velocity. However, these results are likely in part due to the reduced joint motion that is limited by the AFO. This additional confounding factor would hinder our ability to interpret the findings and focus on biological factors that affect energy absorption in the present study. Thus, participants wore AFO were excluded from the final analysis. Nonetheless, given that AFO is commonly worn by individuals with chronic stroke, future research investigating how AFO affects impact absorption and limb loading is warranted.In conclusion, we studied the neuromechanical factors underlying impaired limb loading in individuals with chronic stroke versus able-bodied controls. We found that reduced ankle dorsiflexion velocity and reduced rate of energy absorption are likely key factors that limit rapid limb loading capacity following stroke. In addition, those with reduced rate of energy absorption at the paretic ankle joint revealed by induced limb loading assessment had a prolonged double support duration and decreased gait velocity when walking at maximum speed. Future research will determine the effectiveness of repetitive limb loading perturbation training on improving limb loading, impact absorption, and walking ability post-stroke.


Loneliness is a well-established risk factor for poor mental health. During COVID-19, loneliness and mental health have been exacerbated by widespread disease-related mortality, suggesting that loss of a loved one may influence this relationship. Using data from 187 older adults in Puerto Rico, we assessed the association between loneliness and mental health and the potential moderating role of loss. Moderated multivariable linear regression results indicated that loneliness was significantly, positively associated with mental health . Although loss due to COVID-19 was not significantly associated with mental health (p = .473), it did moderate the relationship between mental health and loneliness . The lack of significant association of loss and mental health contrasts with previous research on COVID-19 and warrants further investigation. Nevertheless, the statistically significant interaction suggests that grief should be considered when assessing individual and community support during the pandemic.


Currently, tumor treatment research still focuses on the cancer cells themselves, but the fact that the immune system plays an important role in inhibiting tumor development cannot be ignored. The activation of the immune system depends on the difference between self and non-self. Unfortunately, cancer is characterized by genetic changes in the host cells that lead to uncontrolled cell proliferation and evade immune surveillance. Cancer immunotherapy aims to coordinate a patient’s immune system to target, fight, and destroy cancer cells without destroying the normal cells. Nevertheless, antitumor immunity driven by the autoimmune system alone may be inadequate for treatment. The development of drug delivery systems (DDS) based on nanoparticles can not only promote immunotherapy but also improve the immunosuppressive tumor microenvironment (ITM), which provides promising strategies for cancer treatment. However, conventional nano drug delivery systems (NDDS) are subject to several limitations in clinical transformation, such as immunogenicity and the potential toxicity risks of the carrier materials, premature drug leakage at off-target sites during circulation and drug load content. In order to address these limitations, this paper reviews the trends and progress of biomimetic NDDS and discusses the applications of each biomimetic system in tumor immunotherapy. Furthermore, we review the various combination immunotherapies based on biomimetic NDDS and key considerations for clinical transformation. Cancer is a major public health problem worldwide and the second leading cause of death in the world . An estiThe advent of immunotherapy has changed the paradigm of cancer treatment . ImmunotIn addition, most cellular immunotherapies have been used primarily in patients with advanced cancer; so, response rates for the less advanced disease have yet to be fully determined . HoweverTo address the limitations of current cancer treatments, this article reviews cancer immunotherapy strategies based on biomimetic nanodrug delivery systems (BNDDS), ranging from antigen/adjuvant delivery vehicles to compounds and their combinations targeting tumor antigen-specific T lymphocytes. In recent years, BNDDS have attracted great attention due to their potential applications in drug delivery A tumor is still considered as one of the most common and serious diseases, which directly damages human health . CompareCancer vaccines can be divided into cells, viral vectors, or molecules  . CellulaImmune checkpoint therapy is a cluster of therapies that modulate the activity of T cells to improve the immune response against tumors . CurrentAmong these, CTLA-4 is highly expressed on the surface of activated T cells and binds to CD80 or CD86, inhibiting T cells from entering S phase from the G1 phase, and thus downregulating or terminating the T cell response . CTLA-4 ACTs use a patient’s natural T cells, or natural killer cells, to detect and eliminate fast-growing tumors . The firMAbs, as effective antitumor tools, have significantly advanced cancer therapy because of their targeting specificity and virtually no “off-target” effects compared to most small molecule drugs . In receCytokines are secreted by various of cell types and are regulators of cell activity . CytokinInterferon-α, which produces anti-tumor effects in several hematological malignancies and solid tumors . In maliWith the development of nanotechnology, various nanoparticle structures have been used as carriers to deliver therapeutic drugs or imaging tags to the site of tumor growth . These nin situ cancer vaccine to stimulate multiple steps of the immune activation process and induce a powerful antitumor immune response  and exogenous substances . Among vin situ, but also modulate the TME, thereby inhibiting tumor growth . This study provided a platform for delivering costimulatory receptor mRNA to immune cells to enhance antitumor immunity.High-density lipoprotein (HDL) is a naturally occurring nanoparticle that is biocompatible, nonimmunogenic, and fully biodegradable . These er growth . Similarr growth  developeCheckpoint block-based immunotherapy has shown unprecedented efficacy in cancer treatment, but its clinical implementation is limited by the low rate of host antitumor response . Recent + T cells and CD8+ T cells) in tumors, thereby effectively inhibiting both primary and distal tumors.Immune responses depend on communication and recognition between immune cells and between immune cells and other cells . Immune In order to overcome the limitations of current cancer treatments, Kang et al. prepared biomimetic nanoparticles using poly (lactic-co-glycolic) acid (PLGA) NPs coated on the T membrane of the EL4 cell line (TCMNP) . Its advPlatelets (PLT) are circulatory guards in the blood stream and have chemotactic effects on damaged vasculature and tumor tissue. Recent studies have shown that PLT membranes can be used as biomimetic coatings 3O4) and masking it with a platelet membrane (Fe3O4-SAS@PLT) could improve the treatment of tumors  to enhance effective iron death sensitivity and improve cancer immunotherapy. In addition, it has been found that Fe3O4-SAS@PLT could not only effectively trigger the iron death of tumor cells by inhibiting the Xc− transport pathway of the glutamate–cystine reverse transport system  into magnetic nanoparticles  laser was used to stimulate the immune system to eliminate residual and metastatic cancer cells.The erythrocyte membrane (EM) is highly biocompatible and has native functional proteins of the original cell . Current3O4-ICG@IRM) for synergistic photothermal immunotherapy of ovarian cancer. Due to the basic properties of the CD47 protein on red blood cells, these carefully synthesized nanoparticles are different from bare Fe3O4-ICG NPs or ID8-CM-coated NPs (Fe3O4-ICG@ID8-CM), can significantly prolong the cycle time, and because cancer cells express surface adhesion molecules with homologous adhesion domains, they can actively target ID8 tumors. In addition, tumor antigens on IRM can also induce an antitumor immune response when Fe3O4-ICG@IRM is phagocytized by the spleen or lymph nodes. Meanwhile, Fe3O4-ICG@IRM NPs not only showed excellent PTT and an in vivo tumor temperature close to 60°C, but they could also further release whole cell tumor antigen through hyperthermo-induced tumor necrosis, enhancing antitumor immunotherapy. Similarly, EM has been investigated as a suitable drug carrier for cancer treatment due to its essential biocompatibility and non-immunogenicity . At the In recent years, immunotherapy has shown good clinical effects, but the low response rate and off-target side effects hinder its wide application. The latest progress made in nanotechnology is expected to improve the efficacy of cancer immunotherapy . HoweverAlthough most reports emphasize the advantages of BNDDS, such DDS also have various limitations. For example, cell membrane coating technology still faces obstacles such as limited therapeutic effect in practical application, mainly due to the complex preparation process and relatively low production . TherefoCurrently, the rapid development of nanotechnology provides a new direction for improving the efficacy of clinical standard models. Cancer immunotherapy has shown efficacy in many cancer types . Howeverin-vivo studies to estimate possible side effects and therapeutic effects. Therefore, the local and systemic toxicity of nanosystems to normal organs and tissues should be carefully studied and evaluated. However, despite these challenges, researchers continue to advance the design of BNDDS and expand their applications.BNDDS are biological vectors inspired by various cell membranes that inherit intrinsic properties of the source cells, including immune escape and tumor tropism . Theoret


According to Haldane’s Rule, the heterogametic sex will show the greatest fitness reduction in a hybrid cross. In birds, where sex is determined by a ZW system, female hybrids are expected to experience lower fitness compared to male hybrids. This pattern has indeed been observed in several bird groups, but it is unknown whether the generality of Haldane’s Rule also extends to the molecular level. First, given the lower fitness of female hybrids, we can expect maternally inherited loci  to show lower introgression rates than biparentally inherited loci  in females. Second, the faster evolution of Z-linked loci compared to autosomal loci and the hemizygosity of the Z-chromosome in females might speed up the accumulation of incompatible alleles on this sex chromosome, resulting in lower introgression rates for Z-linked loci than for autosomal loci. I tested these expectations by conducting a literature review which focused on studies that directly quantified introgression rates for autosomal, sex-linked, and mitochondrial loci. Although most studies reported introgression rates in line with Haldane’s Rule, it remains important to validate these genetic patterns with estimates of hybrid fitness and supporting field observations to rule out alternative explanations. Genomic data provide exciting opportunities to obtain a more fine-grained picture of introgression rates across the genome, which can consequently be linked to ecological and behavioral observations, potentially leading to novel insights into the genetic mechanisms underpinning Haldane’s Rule. An often invoked explanation for Haldane’s Rule relies on dominance theory, which is based on the Dobzhansky–Muller incompatibility model . This thDrosophila where substitutions on the X-chromosome have a larger impact on hybrid sterility and unviability than autosomal substitutions  might lead to lower levels of genetic diversity on the Z-chromosome. The consequent lower effective population size of the Z-chromosome will then render it more susceptible to random processes, such as genetic drift  and indigo bunting (P. cyanea) for autosomal, Z-linked, and mitochondrial loci. The results were in accordance with Haldane’s Rule, showing higher levels of introgression for autosomal loci compared to Z-linked and mitochondrial ones. In general, most of the studies that applied coalescent modeling reported introgression patterns in line with Haldane’s Rule: 6 studies  found higher introgression rates for autosomal loci compared to Z-linked loci, and 8 studies (57%) estimated higher introgression rates for autosomal loci than for mitochondrial loci.Most studies applied coalescent modeling to quantify introgression rates for different genomic markers. This approach allows researchers to calculate introgression rates between populations or species within the framework of an Isolation-with-Migration (IM) model. These models use a Bayesian Markov chain Monte Carlo (MCMC) method to estimate population size, migration rate, and splitting time parameters . By runnTen studies relied on cline analyses to estimate introgression rates for different genomic classes. Cline analyses quantify the changes in allele frequencies occurring along a geographic transect of a hybrid zone. According to cline theory, there is a straightforward relationship between selection against hybrids and cline width, namely that stronger selection against introgression results in a narrower cline . GenomicNm from genetic differentiation statistics, where Nm is the product of the effective population size (N) and the rate of migration (m) among populations (Nm = 0.065) and high nuclear introgression (Nm = 4.9) between the common chiffchaff (Phylloscopus collybita) and the Iberian chiffchaff (P. brehmii). Similarly, introgression of nuclear loci (Nm = 1.1) was higher compared to mitochondrial loci (Nm = 0.11) for greater spotted eagle (Aquila clanga) and lesser spotted eagle (A. pomarina)  . HoweverHippolais icterina) and melodious warbler (H. polyglotta) to different parental and hybrid classes. This assignment exercise revealed introgression of autosomal loci, but indicated no exchange of mitochondrial loci. A similar approach in a hybrid zone between black-capped chickadee (Poecile atricapillus) and Carolina chickadee (P. carolinensis) revealed the same pattern: autosomal introgression, but no mitochondrial introgression  and common nightingale (L. megarhynchos) yielded sterile females and fertile males (Ammodramus caudacutus) and Nelson’s sparrow (A. nelsoni) indicated that hybrid females had lower survival rates than hybrid males  were in accordance with dominance theory as an explanation for Haldane’s Rule, confirming the generality of this pattern on the molecular level. However, just because a molecular pattern is in line with the expectations of Haldane’s Rule does not necessarily mean that reduced introgression of particular loci is due to the lower fitness of female hybrids. Other mechanisms, such as mate choice, local selection pressures, or sex-biased dispersal, can account for these patterns . To confle males . Similarid males . Hence, Geospiza fortis) and the cactus finch (G. scandens) pointed to reduced introgression on the Z-chromosome in comparison with autosomal loci. This pattern might be due to genetic incompatibilities on the Z-chromosome , but detailed field observations showed that particular mating patterns—unrelated to female fertility—could explain the introgression rates (The lack of hybrid fitness estimates is not surprising as this parameter can be extremely difficult and cumbersome to measure in wild populations . Moreoveon rates . This exAnas platyrhynchos) and spot-billed duck (A. zonorhyncha) resulted in higher introgression rates for mitochondrial loci compared to autosomal loci. This pattern can be explained by asymmetrical mating patterns and sex-biased dispersal between these duck species. Male spot-billed ducks tend to disperse more than the philopatric female spot-billed ducks. Hence, male spot-billed ducks are more likely to come into contact with female mallards. Consequently, most hybridization events involved a male spot-billed duck and a female mallard. When the resulting hybrids return to the breeding grounds of spot-billed ducks, they carry the mallard mtDNA with them, leading to asymmetrical introgression of mtDNA from mallards into spot-billed ducks. In contrast, nuclear introgression is mediated by both males and females, so nuclear loci are not expected to reflect dispersal differences between the sexes. In this case, introgression rates for nuclear loci were lower than introgression rates for mitochondrial ones, although still primarily from mallards into spot-billed ducks. This exception to Haldane’s Rule emphasizes the importance of understanding the life history and behavioral characteristics of the study system.Only 2 studies reported introgression patterns that were not in line with Haldane’s Rule. Aegithalos bonvaloti) and sooty tit (A. fuliginosus) compared to negligible exchange of autosomal and Z-linked loci (The second exception concerns high levels of mitochondrial introgression between black-browed tit (ked loci . The autked loci , sometimked loci . In addiked loci . The fixked loci . In someked loci . RegardlLonchura munias based on a limited number of divergent autosomal regions, while mitochondrial introgression was deemed less likely based on the observation of distinct mitochondrial haplotypes. Although these patterns are consistent with Haldane’s Rule, they could also be explained by the higher mutation rates in mtDNA (Despite the increasing availability of genomic data for birds, only one study used a genomic approach to directly estimate introgression rates . The lacin mtDNA  or selecin mtDNA . In addiin mtDNA . Based oA second explanation for the lack of genomic studies concerns the recent focus in speciation genomics on detecting barrier loci that might contribute to reproductive isolation . The comFinally, no study included W-linked loci in the analyses. The W-chromosome is notoriously difficult to sequence due to its haploid nature and high repeat content . HoweverThese knowledge gaps—estimating local introgression rates across the genome and studying the W-chromosome—can be addressed as genomic resources and methods to quantify introgression are constantly improving . A more


Objective. To determine the effect of epilepsy on intracranial electroencephalography (EEG) functional connectivity, and the ability of functional connectivity to localize the seizure onset zone (SOZ), controlling for spatial biases. Approach. We analyzed intracranial EEG data from patients with drug-resistant epilepsy admitted for pre-surgical planning. We calculated intracranial EEG functional networks and determined whether changes in functional connectivity lateralized the SOZ using a spatial subsampling method to control for spatial bias. We developed a ‘spatial null model’ to localize the SOZ electrode using only spatial sampling information, ignoring EEG data. We compared the performance of this spatial null model against models incorporating EEG functional connectivity and interictal spike rates. Main results. About 110 patients were included in the study, although the number of patients differed across analyses. Controlling for spatial sampling, the average connectivity was lower in the SOZ region relative to the same anatomic region in the contralateral hemisphere. A model using intra-hemispheric connectivity accurately lateralized the SOZ (average accuracy 75.5%). A spatial null model incorporating spatial sampling information alone achieved moderate accuracy in classifying SOZ electrodes . A model incorporating intracranial EEG functional connectivity and spike rate data further outperformed this spatial null model . However, a model incorporating functional connectivity without spike rate data did not significantly outperform the null model . Significance. Intracranial EEG functional connectivity is reduced in the SOZ region, and interictal data predict SOZ electrode localization and laterality, however a predictive model incorporating functional connectivity without interictal spike rates did not significantly outperform a spatial null model. We propose constructing a spatial null model to provide an estimate of the pre-implant hypothesis of the SOZ, and to serve as a benchmark for further machine learning algorithms in order to avoid overestimating model performance because of electrode sampling alone. We employed multiple approaches to control for the biases highlighted above, investigating three main questions: (a) What is the effect of epilepsy on intracranial EEG connectivity? (b) How accurately can intracranial EEG functional connectivity localize and lateralize the SOZ? And (c) How can studies of intracranial EEG connectivity control for spatial bias? We found that (a) SOZ electrodes and SOZ regions demonstrate reduced connectivity, (b) reduction in connectivity predicts SOZ laterality, and (c) a spatial null model controlling for the choice of electrode placement estimates the clinicians’ pre-implant hypothesis of seizure generators, and this null model can serve as a benchmark to assess the performance of future predictive models using intracranial EEG.2.2.1.This study was approved by the Hospital of the University of Pennsylvania (HUP) Institutional Review Board. Written informed consent was obtained from each participant.We performed a retrospective analysis of all sequentially participating patients with drug-resistant epilepsy who underwent intracranial EEG recording as part of presurgical planning at HUP from January 2015–October 2021. Electrode targets were chosen according to the clinical pre-implant hypothesis of the seizure generators. In cases of suspected unilateral seizure generators, electrodes were sometimes implanted in the contralateral hemisphere if there was conflicting pre-implantation data .Seizures, SOZ electrodes, and the seizure onset lateralization  were identified by a board-certified epileptologist as part of the clinical evaluation, and confirmed at an epilepsy surgery case conference. Patients without seizures during the recording period were defined to have no SOZ electrodes and no seizure onset lateralization for the purpose of this study.Pre-implant T1-weighted Magnetization-Prepared Rapid Acquisition Gradient Echo (MPRAGE) magnetic resonance imaging (MRI), post-implant T1-weighted MPRAGE MRI, and post-implant computed tomography images were acquired to localize electrodes for both the original and revised implantations. In-house software  was used2.2.The supplemental methods section fully describes our approach to EEG recording, pre-processing, and spike detection. Briefly, we recorded intracranial EEG using a combination of grid, strip, and depth electrodes (including stereo-EEG), according to clinical needs. We performed automated rejection of intracranial EEG time segments detected to have heavy artifact, then applied a common average reference to intracranial EEG signals, and subsequently notch-filtered (removing 60 Hz noise) and bandpass-filtered (1–70 Hz) the resulting signals. We performed automated spike detection using a modified version of a previously published algorithm . We vali2.3.electrodes × Nelectrodes matrix, where Nelectrodes is the number of electrodes, and each i, j element of the matrix represents the (signed) Pearson correlation coefficient between intracranial EEG signals on electrodes i and j. We averaged the functional network across all two-second time windows, yielding one Nelectrodes × Nelectrodes functional network for each one-minute segment. The choice of two-second window and subsequent averaging over longer windows has been used in prior studies to analyze time-varying networks. ) for each additional normalized connectivity unit  Controlling for spatial sampling bias, the SOZ region has lower connectivity to the rest of the brain than contralateral control regions figure , and thi5.1.Restricting analysis to regions with symmetric electrode coverage, the average connectivity to the SOZ region is lower than to the same region in the opposite hemisphere . However, we expect that potential biases in intra-regional electrode numbers will affect both the variance in inter-regional connectivity measurements and any intra-regional connectivity measurements, limiting applicability to potential future analyses.A limitation of the symmetric coverage method we used in our analysis is that it does not control for differences in the 5.2.The average intra-hemispheric connectivity in a network reduced to regions with symmetric electrode coverage correctly lateralized the SOZ 76% of the time, similar to spike rate data figure . The mod5.3.A model using only information about where electrodes were placed exceeded chance  at predicting SOZ electrodes. It is challenging to compare this null result to that of other published analyses because those of which we are aware all predict somewhat different things. Noting that limitation, to put our result into perspective, a study using intracranial EEG networks to identify the epileptogenic zone in patients with good surgical outcome reported an AUC of 0.92 , a studyImportantly, future spatial null models that outperform the one presented here could likely be constructed. Another limitation of the spatial null model is that it itself is subject to bias: clinicians choose electrode placement and they also determine the SOZ electrodes. Future studies could compare a spatial null model against a model informed by intracranial EEG data in their ability to predict surgical outcome, rather than the clinician-defined SOZ electrodes, which would avoid this circularity. Finally, implantation strategies vary across institutions , and therefore the external validity of our proposed spatial null model likely varies across institutions.5.4.not significantly outperform a spatial null model at localizing the SOZ electrodes. This negative result is somewhat limited by the conservative bootstrap analysis, but it suggests that, while reduced functional connectivity likely localizes the SOZ electrodes, its independent predictive effect is small in our study. We do not claim that functional connectivity more generally does not predict the SOZ electrodes. Given that the scope of this study was to examine and control for spatial biases in intracranial EEG network analysis rather than to optimize a SOZ predictor, we did not exhaustively examine other features  that may outperform the choices in this study [A model incorporating functional connectivity and spike rates outperforms a spatial null model at localizing the SOZ electrodes, providing additional evidence that interictal data can help identify seizure generators , 14, 34.is study . Regardl6.Network analysis of interictal intracranial EEG is a promising approach to improve our understanding and treatment of epilepsy, but is prone to spatial bias. We employed multiple approaches to control for spatial bias. We found that (a) functional connectivity is reduced in the SOZ region and accurately lateralizes the SOZ, (b) a model using only spatial sampling, ignorant of intracranial EEG data, achieves moderate performance in localizing the SOZ electrodes, and (c) a model incorporating interictal EEG biomarkers outperforms a model using spatial sampling information alone. Constructing a spatial null model is a useful method to assess how well we can predict seizure generators prior to ever examining intracranial EEG data, and can serve as a benchmark against which to compare performance of machine learning algorithms using intracranial EEG data.


Marfan syndrome (MFS) is a systemic disorder of connective tissues caused by insufficient elastic fiber formation that leads to structural weakness and results in various tissue disorders, including cardiovascular and periodontal disease. Notably however, the risk of periodontal disease in MFS patients affected by an aortic aneurysm or dissection has not yet been clarified.We investigated the periodontal condition in the following three groups: MFS patients diagnosed with an aortic aneurysm or dissection with a planned aortic surgery (MFS surgery), MFS patients who had already undergone aortic surgery (MFS post-surgery) and healthy control patients . The periodontal condition of all of these patients was evaluated at their first visit, reassessed again at two-month after the first visit, and evaluated again at a six-month follow-up after the reassessment.A total of 14 participants, 3 MFS surgery patients, 4 MFS post-surgery patients and 7 healthy control volunteers were examined. Saliva examinations revealed no significant differences between any of the groups at the first visit, reassessment, or follow-up. Interestingly, the MFS surgery cases showed a higher BOP and PISA at the first visit and follow-up compared with the other groups. In contrast, the MFS surgery patients showed an improvement in their LVDd and EF values, both markers of cardiac function, at the reassessment and follow-up compared with the first visit.MFS associated with an aortic aneurysm or dissection leads to a higher risk of periodontal disease, indicating the need for more frequent oral hygiene maintenance in these patients. In addition, MFS patients who undergo frequent professional cleaning of their teeth show a lower onset of cardiovascular disease, suggesting that professional oral hygiene in these cases contributes to a healthier condition.The online version contains supplementary material available at 10.1186/s12903-022-02361-5. Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue that affects approximately 1 in 5000 people. It is caused by missense mutations of Fibrillin-1 (FBN-1), a component of the extracellular microfibrils, leading to cardiovascular complications including aortic root dilatation, dissection, and rupture which are the leading causes of morbidity and mortality . Blood vPeriodontal disease is caused by a bacterial infection that activates the innate immune response via Toll-like receptors, resulting in the up-regulation of innate immunity cytokines such as Tumor Necrosis Factor-α(TNF-α), Interleukin-1(IL-1), and Interleukin-6(IL-6) to ultimately cause progressive tissue destruction . MFS hasIn contrast to the findings of these aforementioned studies, another clinical study reported that MFS cases did not show high susceptibility to periodontal disease but showed more inflammation of periodontal tissue . HoweverThe present study was approved by the Ethical Committee of Tohoku University in accordance with the Declaration of Helsinki (No. 2018-3-025) and was designed as a case control investigation. The case subjects were MFS patients for whom an aortic surgery had been scheduled (MFS surgery), or who were followed-up after aortic surgery (MFS post-surgery). Patients with no systemic disease including cardiovascular disease, were employed as a healthy volunteer . All the study subjects were ensured not having active caries lesions prior to start this study. MFS patients who were diagnosed in accordance with the established clinical criteria for this disorder  were recAll of the study participants underwent a comprehensive periodontal examination, including oral photography (9 sheets method) and a plaque control record. Probing pocket depth (PPD) and bleeding on probing (BOP) tests were conducted at six sites per tooth  to calculate the periodontal inflamed surface area (PISA), a useful index of the inflammation status for clinical and epidemiological assessments . We alsoAll measurements, including periodontal examinations, saliva tests and oral photographs, were carried out by an experienced dental hygienist at the first visit. Within two months after this first visit, initial periodontal treatments including scaling and root planning were performed. The effects of the initial periodontal treatments were evaluated by periodontal examination after initial periodontal treatment as a reassessment. All of the study subjects visited clinics every two months for supportive periodontal therapy (SPT) after reassessment, their periodontal condition was evaluated after six months as a follow-up, and these treatments were continued as maintenance therapy Fig. . AdequatAll of the study subjects underwent an initial periodontal treatment, including scaling and root planing after their first visit for two months. Reassessments were carried out at two months after the first visit. Supportive periodontal therapy was performed every two months after the reassessment. Follow-up examinations were carried out at six months after the reassessment. All of the study participants underwent periodontal examinations via saliva tests and oral photographs at the first visit, reassessment and follow-up.Cardiac function was evaluated by assessing the left ventricular end-diastolic diameter (LVDd) and ejection fraction (EF) in the surgery and post-surgery MFS patients.The recorded data were documented and analyzed using the data procession program JMP Pro 15 . Each individual subject counted as a statistical unit in all tests. The mean values and range were calculated for all parameters. The Tukey–Kramer method was applied to determine significant differences between the groups. For all tests, a P-value of ≤ 0.05 was considered statistically significant.A total of 14 participants 3 MFS surgery patients ; 4 MFS post-surgery patients , and 7 healthy volunteers , were included in this study  and in Patient 2 at the first visit (arrowhead).We next evaluated the cardiac function status during periodontal treatment at the first visit, reassessment, and follow-up period in the MFS surgery and post-surgery groups using the established LVDd and EF indicators Table . All of Prevotella intermedia were significantly lower in MFS patients with cardiovascular disease compared to the non-MFS cardiovascular disease cases [We here provide the first report that has compared periodontal disease activity between MFS surgery, MFS post-surgery, and healthy control subjects during a long-term observation period to further investigate whether MFS increases the risk of periodontal disease. Although it was difficult to recruit suitable subjects due to the rare prevalence of MFS in this study, our results suggested that the periodontal disease status worsened in the MFS surgery patients after a prolonged gap in follow-up maintenance due to the COVID-19 pandemic. From these findings, we proposed that professional intervention decreases the onset of periodontal disease activity in MFS patients. However, more frequent maintenance was required in MFS patients who received surgery to treat aortic aneurysm and dissection. In addition, the cardiovascular function was unaffected during treatment for oral issues in the MFS surgery and post-surgery groups, suggesting that professional intervention may contribute to a more stable health condition in these patients. Major cardiovascular manifestation of MFS can include a thoracic aortic aneurysm, mitral valve prolapse (MVP) and cardiac dysfunction . The repse cases . In addise cases . In our se cases . AnotherThe management of MFS patients who develop aortic aneurysms includes medical therapy and prophylactic surgery to reduce the risk of an aortic dissection , 21. HowProfessional oral hygiene prevents the risk of periodontal disease in MFS patients diagnosed with cardiovascular disease and regular examinations are required in these cases where an aortic surgery is planned to reduce the risk of periodontal disease. In addition, none of the MFS patients analyzed in this study showed a progression of their cardiovascular disease, suggesting that a professional approach to oral hygiene contributes to maintaining a healthy condition in these cases.Additional file 1. Supplementary Table 1. Results from buffer capacity, S.mutans, and Lactobacillus testing.


The global cancer cases and mortality rates are increasing and demand efficient biomarkers for accurate screening, detection, diagnosis, and prognosis. Recent studies have demonstrated that variations in epigenetic mechanisms like aberrant promoter methylation, altered histone modification and mutations in ATP-dependent chromatin remodelling complexes play an important role in the development of carcinogenic events. However, the influence of other epigenetic alterations in various cancers was confirmed with evolving research and the emergence of high throughput technologies. Therefore, alterations in epigenetic marks may have clinical utility as potential biomarkers for early cancer detection and diagnosis. In this review, an outline of the key epigenetic mechanism(s), and their deregulation in cancer etiology have been discussed to decipher the future prospects in cancer therapeutics including precision medicine. Also, this review attempts to highlight the gaps in epigenetic drug development with emphasis on integrative analysis of epigenetic biomarkers to establish minimally non-invasive biomarkers with clinical applications. Cancer is a multifactorial disease developed as a result of several genetic as well as epigenetic changes. Epigenetics is a process that involves the alteration of gene expression without changing the DNA sequence. It is a Greek word meaning above or over the genome, which was coined by Conard Waddington in 1942 . The proResearch over a decade has focused on promoter DNA methylation and histone modifications as the two main molecular mechanisms that mediate the process of epigenetic regulation in anticancer therapies and biomarker discovery. Studies have demonstrated that the altered DNA methylation genes or patterns can be potentially used as biomarkers for proper cancer screening, diagnosis, and prognosis . Thus, eIn addition, the tumour microenvironment (TME) of the cancer cells contains aberrant epigenetic marks which are known to cause a favorable environment for tumor growth . The exiSeveral molecular mechanisms exist behind epigenetic regulation, including DNA and RNA methylation, histone modifications, and ATP dependent nucleosome remodelling which have been discussed here.via transcriptional suppression , including DNMT1, DNMT3a, and DNMT3b . These epression . On the pression . The glopression . Thus, ipression . In factpression . In addipression . Even, tpression .6-methyladenosine (m6A) modification is the most abundant and it is recognized by RNA binding proteins that affect many characteristics of mRNA function , histone methyltransferases (HMTs), and histone kinases. These epigenetic marks are known as writers and act as transcriptional co-activators. On the contrary, erasers  function as transcriptional co-repressors by removing these groups from histone end  through chromatin remodelling complexes by ATP hydrolysis. These chromatin-remodelling complexes can be classified into switching defective/sucrose nonfermenting (SWI/SNF), chromodomain-helicase DNA-binding protein (CHD), imitation SWI (ISWI), and INOsitol requiring mutant 80 (INO80) complexes. The catalytic subunit of these complexes performs DNA translocation along with the histone core of the nucleosome .The SWI/SNF complexes are one of the most widely studied ATP dependent chromatin remodelling complexes. They are found to be mutated in 25% of human cancers  and are The next important complex is ISWI which mobilizes nucleosomes by helping the transcription factors to bind a nucleosome-free DNA. Unlike SWI/SNF complex, ISWI is held to nucleosomes by a SANT and a SLIDE domain . As repoEpigenetic changes like DNA methylation and histone modification detected in early tumorigenesis and cancer progression have been proposed as biomarkers for early cancer detection, tumor prognosis, and treatment response . They arProstate cancer is the second most common cancer (14.8%) in men and the fourth leading cause of cancer related death (6.6%) , globallFew studies have reported a correlation of promoter methylation at the methylguanine-DNA methyltransferase (MGMT) gene with favorable treatment outcomes in glioblastoma patients treated with temozolamide, suggesting its possibility to be used as an epigenetic biomarker .Studies have shown that high levels of hypermethylated DNA exists in colorectal cancer which leads to genomic instability . The metp = 0.0001)  .In 2021 , the incSeveral studies have suggested alterations in histone-modifying enzymes like enhancer of zeste homolog 2(EZH2). This enzyme is encoded by EZH2 gene, which participates in histone-methylation and transcriptional repression. EZH2 has significantly reduced expression of histone in breast cancer . AnotherStirazaker et al., 2015 divided TNBCs into different epigenetics groups viz. high, intermediate, and low-risk groups based on epigenetic subtypes and the methylated region that is correlated with the progression of the disease. One of the recent studies has also confirmed a correlation between shorter periods of reduction in analysed TNBC-samples  and hypeThe worldwide incidence and mortality rates of ovarian cancer in the year 2021 were 1.1% and 2.3%, respectively . HistoneResearch has recognized the role of epigenetics in developing drug resistance thereby affecting cancer treatment. Cacan et al. (2016) reported that the loss of apoptosis antigen 1 expression impacts drug resistance, which is mediated by histone deacetylase 1 (HDAC1) in chemoresistant ovarian cancer cells . Using CA recent study has described hypomethylation of developmental genes MSX1, DAXX and TMEM88. mRNA expression of these developmental genes is associated with platinum resistance and inversely correlated with promoter methylation in ovarian cancer patients by treatment with Guadecitabine (DNA methyl transferase inhibitor) and cisplatin ..DNA methylation regulates epithelial mesenchymal transition (EMT) by lncRNA (HOTAIR) and it is a sign of resistance to carboplatin . A studyQuite a few epidrugs are approved for the treatment of several cancers. These epidrugs are the inhibitors of DNA methyltransferase (DNMTi) and histone deacetylase (HDACi) enzymes . The firOne of the main problems in the application of epidrugs is that the drug binds to other targets rather than its own target. This is called “off-target effects” in epigenetic therapy. Growing epi-research has shown that the use of synthetic lethal approaches might result in apoptosis. In this approach, two inactive genes which synergistically result in apoptosis are selected and combined. These epidrugs are delivered to target synthetic lethal partners having genetic mutations in cancer cells. However, these drugs are less/not toxic to non-cancerous cells with no mutations, resulting in more precise therapy. One such epidrug is the inhibitor of the histone methyltransferase DOT1L (disrupter of telomere silencing 1-like), Pinometostat which specifically kills the MLL-fusion leukaemia cells . Another®” assay has received the Conformité Européenne (CE) mark as In Vitro Diagnostic (IVD) test for lung cancer diagnosis. It is based on methylation analysis of SHOX2 (Short Stature Homeobox 2) and PTGER4 (the prostaglandin E receptor 4) genes  has also approved non-invasive DNA methylation tests of these genes for early colorectal cancer screening programmes  genes . Numerouogrammes .Another CE-IVD marked test, miRpredX-31-3p kit  is based on the quantification of miR-31-3p expression levels. It is used to recognise metastatic colorectal cancer patients who can benefit from anti-EGFR  therapy . Thus, aThe future of epidrug development involves the use of integrated network medicine with epigenetics, where several analytical methods like protein-protein interaction (PPI) networks, correlation-based networks and gene regulatory networks are utilized to roll out key genes, relevant regulatory and co-regulatory networks in causing disease pathogenesis . A groupZheng et al. (2020), used deep neural network (DNN) algorithm to predict cancer diagnosis in the DNA methylation data of 7,339 patients of 18 different cancer origins from The Cancer Genome Atlas (TCGA) . RecentlThus, network medicine might advance the field of epigenomics as it is possible to rule out the co-regulatory networks of DNA methylation . HoweverEpidrug development is accompanied by its own set of challenges that needs to be addressed for the establishment of locus-specific, highly sensitive and cost-effective biomarkers. The main obstacle is to comprehend the “casualty” of epigenetics, meaning that whether the epigenetic abnormality is a result of malignancy or malignancy itself is caused due to these variations. It is crucial to decipher the link between epigenetic differences and cancer progression to establish a biomarker with potential utility in the cancer clinics. This is only possible by pursuing special cohort studies where epigenome profiling can be maintained before the start and after the end of the disease. The high cost of such studies limits their application.Another challenge is the lack of locus specificity which might cause epigenome-wide “off-target” effects leading to the loss of important gene function and can be resolved using new epigenome editing approaches . With thIt is understandable that the epigenetic changes, especially the methylation patterns are very informative in establishing both diagnostic and prognostic biomarkers. But the problem lies in the complex assay systems, imprecise reproducibility, inadequate clinical validation, and false discovery of these biomarkers . TherefoIt is well established that there is a link between cancer and epigenetics. Some epigenetic drugs have already been approved by US-FDA and many more epidrugs are under development for appropriate cancer detection and treatment. Furthermore, there is a scope for epigenetics-based cancer therapies delineating the tumor heterogeneity in different cancers with precision, that should focus on cell-cell behaviour in TME. In addition, epigenetic research should focus on network and precision medicine approaches for the discovery of novel biomarkers so that they can be safely translated to the clinic after proper clinical trials. Thus, proper identification of the epigenetic landscape behind the cancer progression and establishing therapeutic drugs is the future of epigenetics in cancer without forgetting to overcome the challenges faced in effective epidrug development.