Determinants of elevational distribution of butterfly species richness and abundance in the tropics are poorly understood. Here we assess the combined effects, both additive and interactive, of seasonality and habitat structure on the elevational distribution of butterflies in the Uluguru Mountains, Tanzania. We sampled butterflies along a 1100 m elevational gradient that extended from 1540 to 2639 m using a time-constrained fixed-area method during the short to long rains and long to short rains transitions, and in habitat structure classified as closed or open. We used semi-parametric generalized linear mixed models to assess the relation between butterfly species richness or abundance, and seasonality, habitat structure, family and elevation. For all species combined, species richness declined with elevation in both open and closed habitats during the long to short rains transition. During the short to long rains transition, species richness displayed a mid-elevation peak across habitats. Among the three focal families  similar patterns in the elevational distribution of species richness were observed. Species abundance declined or remained stable with elevation across seasons and habitat structure; the exception being species abundance in open habitat during the short to long rain transition and increased slightly with elevation. Abundance by family did not vary significantly by habitat structure or season. Our results indicate that seasonality and habitat structure shape species richness and abundance of butterflies along an elevational gradient in the Uluguru Mountains. These patterns are important for informing conservation actions because temperature as well as annual and seasonal variation in precipitation are predicted to increase in East Africa as a result of climate change, important determinants of seasonality, while habitat disturbance may increase due to a projected doubling in Tanzania’s population over the next 27 years. Elevational distribution patterns of species can provide valuable insights into interactions between organisms and environmental gradients processes driving those patterns , and knoA number of factors have been proposed as drivers of elevational distribution of species. McCain and Grytnes  have groMost studies of the elevational distribution of insects have been conducted in temperate regions. In contrast, comparatively fewer studies have been conducted in the tropics although the vast majority of species occur here. Yet, even among the better-studied insect taxa in the tropics such as butterflies , 13 therBoth abiotic and biotic factors influence species richness and abundance of tropical Lepidoptera along elevational gradients. Of these factors seasonality , 15 and Seasonality is an important determinant of resource availability and activity patterns in Lepidoptera  and for Habitat structure has also been shown to influence elevational patterns of species richness and abundance in Afrotropical Lepidoptera , 30. PatHere we examine the combined influence of seasonality and habitat structure on the elevational distribution of butterfly species richness and abundance in the Uluguru Mountains, an Afrotropical biodiversity hotspot. Specifically, we compare the influence of the short to long rains transition versus the long to short rains transition (season), closed versus open habitat structure, family (see Study groups in Methods) and their interactions on the elevational distribution of butterfly species richness and abundance. Based on associations reported from non-elevational studies of tropical butterfly species richness, abundance and seasonality and habitat structure , 28, 29 The study was conducted in the Uluguru Nature Reserve in east-central Tanzania . The UluPrecipitation in the Uluguru Mountains is bimodal  with theMean daily temperature also varies seasonally with the short to long rains transition coinciding with the hot season and the long to short rains transition coinciding with cool season . The recWe conducted butterfly sampling along a 1100 m elevational gradient that extended from 1540 m to 2639 m . This grSampling was conducted along two transects that followed established trails in the two largest forest blocks in the northern  and southern  sections of the Uluguru Nature Reserve . Thus, iWe sampled butterflies along the two transects at an interval of ~ 40 m in elevation (range 30 m– 50 m). This interval was selected to permit a fine-scale assessment of the influence of seasonality and habitat structure on the elevational distribution of butterfly species. Prior to initiating sampling and to ensure an approximate equal difference in elevation between adjacent sampling locations, the location for all sampling locations along the transect was defined by waypoints  extracteBetween February 2019 and September 2020, we sampled butterflies at each of the 28 sampling locations on 20 occasions over two seasons. At each sampling location, 10 replicated samples were conducted during both the short to long rains transition (January through February) and long to short rains transition (June through September). We selected these seasons, which follow Maicher et al , to permAt each sampling location along the transect, we sampled butterflies for 20 minutes in a 5 m × 5 m × 5 m plot centered over the trail through visual observations and sweep-net inspections of species we could not identify in the air following sampling protocols outlined in Caldas and Robbins . We seleIn addition, we placed a baited Van Someren trap  at each We classified habitat structure at each sampling location as either closed  or open  based on mean canopy closure. At each sampling location, we recorded four measurements of canopy closure using a densitometer  held at Podocarpus milanjianus, Ocotea usambarensis, Afrocrania volkensii, Ficalhoa laurifola, and Cussonia spicata. The common shrubs are Mostuea brunonis, Chassalia parviflora, Chassalia violacea, Lasiodiscus usambarensis, Galineria coffeoides, Memecyclon mytrilloides, Erthrococca usambarica, Euphorbia usambarica, and Bridelia brideliffolia while the common woody herbs include Crassocephalum manni, Conyza newii, Vernonia adoensis and Ensete ulugurense [Pócs  has publugurense .Mostuea brunonis, Chassalia parviflora, Chassalia violacea, Lasiodiscus usambarensis, Galineria coffeoides, Memecyclon mytrilloides, Erthrococca usambarica, Euphorbia usambarica, and Bridelia brideliffolia, woody herbs include Crassocephalum manni, Conyza newii, Vernonia adoensis and Ensete ulugurense, and grasses and herbs comprised of Hyparrhenia rufa, H. diplandra, Brachiaria brizantha, Bekeropsis uniseta, Arthraxon quartinianus, Cleistachne sorghoides, Polygala macrostigma, and Habenaria splendens [Between 1500–2300 m, sites we classified as open are dominated largely by non-canopy forming tree species and a combination of shrubs including plendens .Pycreus nigricans is the dominant species and can reach heights of up to 5 m. In subalpine grassland the dominant species of grass are Panicum lukwangulense, Andropogon amethystinus, Agrostis kilimandscharica, and Pteridium aquilinum, and herbs and forbs are Geranimum vagans, Helichrysum cymosum, H. abietinum, Senecio cyaneus, Lobelia holstii, and Blaeria johannnis combined with a few scattered trees and shrubs comprising of Myrica salicifolia, Adendocarpus manni, and Berberis holstii [Between 2300–2664 m in the southern Uluguru Mountains sites we classified as open are defined by Pócs  as moorl holstii .Previous analyses of tropical butterfly species richness along elevational gradients have revealed variation in patterns of species richness among taxonomic families  and withThe statistical analyses involved two response variables, species richness (number of species) and species abundance , which were regressed separately on four predictor variables: elevation, season, habitat structure, and family, and all their possible interactions. Season (short to long rains transition and long to short rains transition) and habitat structure (closed and open) each had two levels, while families had three levels .We first modelled the variation in species richness (number of species recorded within each 5 x 5 x 5 m plot per 246.6 sampling-hours) along an elevational gradient using a semi-parametric generalized linear mixed model  and included habitat structure, and family, and all their interactions as factor-type covariates. The fixed part of the model consisted of parametric fixed effects whereas the random part contained non-parametric smoothed effects, hence the name semi-parametric model. The full model was fitted in the GLIMMIX Procedure in the SAS Software  and Fishseasons × habitats, seasons × families, habitats × families, seasons × habitats × families. The ninth variance component is the scale (dispersion) parameter of the negative binomial distribution.The parametric fixed effects were estimated for season, habitat structure, family and all their two- and three-way interactions. The random part of the mixed model consists of nine random effects or variance components. Of these, eight are continuous random effects that perform penalized spline smoothing of the distribution of species richness along elevation. The first captures patterns in the trend in species richness across the elevational gradient common to all the species records, the second to the seventh, respectively, capture patterns in the trend across the elevational gradient common to both seasons, both habitat types, all the three families, A tenth variance component was included to capture spatial autocorrelation in the residuals, represented in terms of the spatial generalization of the first-order autoregressive error structure and assuming an exponentially decaying correlation with distance of separation between observation points along the transect. But this component was not well supported by the data and resulted in an infinite likelihood, likely because it is confounded with the scale parameter of the negative binomial distribution. Thus, the model without spatially autocorrelated residuals adequately accounted for spatial autocorrelation.habitat × families approximated significance, we decomposed this interaction into its simple effect slices. Additionally, the adjusted means and their 95% confidence limits and pairwise differences were tested for significance, after adjusting for multiplicity using simulation adjustment. Predicted species richness and its 95% confidence limits were back transformed to the original count scale using the inverse link (log) function. Residual diagnostics  were used to check model fit, specifically the conditional raw residuals, conditional studentized residuals, and conditional Pearson residuals. The denominator degrees of freedom of the Wald-type F-tests of the fixed effects were adjusted for small sample size using the Kenward-Roger method [The penalized cubic basis splines used with each continuous random effect has 10 equally spaced interior knots plus three knots at the start and three at the end of the observed values of elevation. Because the effect of r method . The samr = 0.91, P < 0.001, Due to the possibility of incomplete sampling of species richness, we estimated species richness with the abundance-based bias-corrected Chao1 estimator in Excel. Because observed and predicted species richness were highly correlated  species richness displayed a hump-shaped mid-elevational peak (~2000 m) in both closed and open habitats during the short to long rains transition while species richness in the family Pieridae increased with elevation in both closed and open habitats in both seasons . ButterfButterfly abundance either declined or changed little along an elevational gradient by season and habitat structure . The excFor two of the three families  the elevational distribution of butterfly abundance displayed a hump-shaped distribution at low to mid elevations (~ 1700 m– 2000 m) in open habitat during both the long to short rains and short to long rains transitions; and in closed habitat during the short to long rains transition . ButterfOur results indicate that elevational patterns of butterfly species richness and abundance are influenced by both seasonality and habitat structure in the Uluguru Mountains. We observed higher species richness and abundance along an elevational gradient in the Uluguru Mountains during the short to long rains transition than during the long to short rains transition. We also observed higher species richness and abundance along the same elevational gradient in open than in closed habitat. However, we did not detect a significant interaction between season and habitat structure along an elevational transect for either butterfly species richness or abundance indicating that the effects of these predictor variables on butterfly species richness and abundance were additive rather than interactive along an elevational gradient in the Uluguru Mountains.The influence of seasonality on elevational patterns of butterfly species richness and abundance in the Uluguru Mountains is consistent with findings from Mount Cameroon in west Africa . MaicherThe seasonal peak in butterfly species richness and abundance in the Uluguru Mountains during the short to long rains transition is also consistent with seasonal patterns of butterfly species richness and abundance in the Kibale forest in Uganda although the annual timing of this seasonal peak differs between these two locations. In the mid-elevation (1100 m– 1590 m) Kibale forest, long-term monitoring of butterflies over a 132 month-period revealed bi-annual peaks in species richness and abundance that occur 3–4 months after the peak in the short  and long rains (termed “large” rains) and corresponding to 2–3 months after the peak in vegetation greenness. However, in the Kibale forest the “small” rains occur between March-May, which coincides temporally with the long rains in the Uluguru Mountains. While the “large” rains in Kibale forest occur between September–November, which coincides temporally with the short rains in the Uluguru Mountains. Yet, both in the Uluguru Mountains and to a lesser degree in the Kibale forest butterfly species richness and abundance is higher during the short to long rains transition than during the long to short rains transition  displayed a hump-shaped mid-elevational peak at ~2000 m in both closed and open habitats during the short to long rains transition while species richness in the family Pieridae increased with elevation in both closed and open habitats in both seasons. On the other hand, butterfly abundance by family did not vary by habitat nor season.Elevational patterns of species richness of butterfly families in the Uluguru Mountains also differ from that reported for these same families along an elevational transect that extended from 117 m to 3104 m in the Sierra de Juárez in southern Mexico  where a Over the last two centuries 77% of the original forest cover in the Eastern Arc Mountains have been lost . The remGlobal climate change is predicted to result in upslope shifts in the elevational range of many species . IncreasS1 Data(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 FigAir temperature was recorded with loggers at 1-h intervals at 1663, 1836, 2004, 2159, and 2588 m over a 14-month period between August 2019 and September 2020. The straight line is described by the following equation: y = 25.1–0.0055 (x).(TIF)Click here for additional data file.S2 FigPredicted species richness was calculated with the abundance-based bias-corrected Chao1 estimator.(TIF)Click here for additional data file.


Colloids are essential materials for modern inkjet printing and coating technology. For printing and coating, it is desirable to have a high density of colloids with uniformity. Binary colloids, which consist of different size colloidal particles, have the potential to achieve high coating density and uniformity from size effects. We report a strategy to attain high-density deposits of binary colloids with uniform, crack-free, and symmetric deposits through droplet evaporation on micropillar arrays. We modify surfaces of micropillar arrays with plasma treatment to control their surface energy and investigate how binary colloidal fluids turn into well-controlled deposits during evaporation with X-ray microscopic and tomographic characterizations. We attribute temporary surface energy modification of micropillar arrays to the well-controlled high-density final deposits. This simple, low-cost, and scalable strategy would provide a viable way to get high-quality, high-density deposits of colloids for various applications. Besides the economic advantage of inkjet printing, additional benefits come from the easy control of inkjet materials, delivering exact quantities of nanoinks through single ink droplets on pre-fabricated substrate patterns, and rendering specific structures and properties7. From a physical viewpoint, colloidal suspensions, including insoluble colloidal particles with diameters of approximately 1 nm to several 9. The drying process of colloidal nanoinks is critical in achieving uniform deposition of colloidal particles but challenging in industrial applications because of coffee-ring effect, characterized by solute segregation at the contact line of evaporating colloidal droplets10, and crack formation, which is induced by air invasion into the deposits12. Both results cause nonuniform and defective deposition of colloids and eventually degrade the electrical and optical properties of deposits. Additionally, the evaporation of monodispersed colloidal nanoinks inevitably causes the generation of empty spaces among spherical colloidal particles13. The maximum packing density from monodispersed spherical particles is 74% when particles are arranged with face-centered or hexagonal closed packing, a crystalline state. The maximum packing density of a monodisperse particles with random packing is 64%14. In binary colloidal particles with two different diameters, small particles are expected to fill in empty space among large particles, increasing the packing fraction and generating a more physically stable deposition16. The increment of packing fraction by using bimodal particles, which leads to highly-dense deposits in the inkjet printing technique, has been shown to enhance electrical and sensing properties of printed devices effectively19. Finding appropriate deposition condition of binary colloids is crucial to improve coating density and uniformity of colloidal nanoinks deposits.Colloidal suspensions that consist of insoluble colloidal particles in a solvent are the main component of inkjet printing, a remarkably economical technique used for device manufacturing in several industries 21, including a micropillar-guided deposition9. Due to surface roughness, hydrophobicity of substrates26 would induce the self-pinning of colloidal fluids on micropillar substrates. A constant contact angle mode (CCA) during droplet evaporation would cause preferential accumulation of colloidal particles at the droplet center and eventually bump formation, as demonstrated in Fig. 28 and can induce the depinning of colloidal droplets on micropillar substrates, as demonstrated in Fig. Achieving uniform deposition of monodispersed colloids through evaporation is essential in recent studiesThis study demonstrates how to achieve symmetrically uniform crack-free hexagonal dense deposits of binary colloids using a surface-modified micropillar-guided deposition. Using high-resolution X-ray microtomography, we measure real-time dynamics of evaporation and deposition processes for binary colloidal droplets on plasma-modified micropillar substrates. Our findings contributes to identify the optimal conditions that are feasible in inkjet printing technology.29. For the wettability control, the surface energy was increased by applying oxygen plasma treatment27. The plasma surface activation process was carried out with a Basic Plasma Cleaner  at a radio frequency (RF) power setting of low (7.2W). The plasma treatment time (We examined two kinds of PDMS (polydimethylsiloxane) substrates for comparison: PDMS with flat surfaces and PDMS with micropillar patterns. The PDMS substrates were fabricated with SYLGARD31. Their surface chemistry is well controlled to be completely immersed and uniformly dispersed in solvents by the supplier. The basic knowledge taken from model particles would be useful in inkjet printing, despite differences in surface chemistry and ink formulation details. Overcoming a limit of monodispersed colloids, binary colloids with significantly different diameters would contribute to dense packing  images through microtomography. The experiments were carried out at the 6C Bio Medical Imaging (BMI) beamline established in the Pohang Light Source (PLS-II), where the beam is composed of monochromatic synchrotron X-rays with 20 keV energy. A We successfully achieve highly uniform and dense deposits of binary colloids on surface-modified micropillar substrates, as described below.The initial depinning dynamics, as illustrated in Fig. 37. Original hydrophilicity is favorable for initial depinning, contributing to flattening the upper bump. Late recovery from hydrophilicity to hydrophobicity is favorable for re-pinning, minimizing the final deposit surface area.The late re-pinning dynamics, as illustrated in Fig. 34. However, the air exposure allows the surrounding substrate to be prone to induce the aging effect, facilitating the recovery dynamics. The recovery dynamics of micropillar substrates are responsible for the late re-pinning dynamics and thus the final minimum deposit surface area.The recovery dynamics of oxygen plasma treatment by aging effects is due to air exposure under ambient conditions  of the deposit due to the highly constricted surface area of the hexagonal prism base (S)  Fig. A, left. (S) Fig. B, left ient Fig. A, right.ent Fig. B, right.9, where the hexagonal deposit volume is obtained as S and H, are quantified in our experiments in (Fig. H is taken on average from the side-view X-ray imaging, for instance, S is taken by subtracting the total surface areas of the pillar tops inside the hexagonal prism from the surface area of the hexagonal prism base, for instance, 9.Hexagonal deposit patterns with geometrical uniformity can be expected by the hexagonal prism model in Fig. . H is tamentclass2pt{minim39.The symmetric deposition is achieved by initial symmetric pinning, as illustrated in Fig. 15, gravity can induce percolation of small particles, leading size separation (Fig. 16. Concerning the mixing volume ratio 40. Furthermore, a mixture with 16. However, the micropillar array hydrophobicity allied to the presence of larger particles would lead to an enhanced movement of smaller particles outwards to the drying interface, constraining the larger particles to stack at the center41. The size separation at The optimum mixtures in binary colloids are crucial to achieve highly-dense uniform patterns. The colloidal suspension, being a binary mixture of particles, has two main criteria: particle size ratio (16. Theoretically, a reachable final packing density is known as 15, while the high-density deposits are achieved as 9. Therefore, binary colloids are more favorable for a high packing density than monodispersed colloids. Our finding on the deposition dynamics will be relevant to the evaporation dynamics of binary colloids, for instance, regarding evaporation dynamics on superhydrophobic surfaces42. Further studies are required to understand evaporation dynamics and to overcome hydrodynamic effects during evaporation to increase deposit densities.Finally, we discuss packing fraction differences before and after plasma treatment. Our results indicate that the higher packing fraction is present for the uniform deposit pattern, consistent with the observation in Fig. 45. This strategy would help to optimize inkjet printing by proceeding with inexpensive, easy-fabricated, well-controlled high-density deposits of binary colloids for many possible applications, being nearly universal.Our study represents a feasible method to achieve high-quality, high-density hexagonal deposits of binary colloids on hexagonal-patterned micropillar substrates. Temporarily turning the substrates hydrophilic with oxygen plasma treatment helps to induce the initial depinning dynamics. The late recovery to hydrophobicity is helpful to induce the re-pinning dynamics of binary colloidal fluids on the substrates. These controlled dynamics would be crucial to achieve symmetric high-density deposits of binary colloids. Additionally, the proposed method forms horizontally uniform heights by removing height differences between the center and the edge of final deposition patterns when using binary colloids and micropillar patterns. The achievable increased packing fraction imply numerous advantages for industrial performancesSupplementary Information.


Background: Recent evidence supports that restoration of the pre-arthritic condition via total knee arthroplasty (TKA) is associated with improved post-TKA performance and patient satisfaction. However, whether the restored pre-arthritic joint line simulates the native mid-flexion biomechanics remains unclear. Objective: We performed a matched-pair cadaveric study to explore whether restoration of the joint line via kinematically aligned (KA) TKA reproduced native knee biomechanics more accurately than the altered joint line associated with mechanically aligned (MA) TKA. Methods: Sixteen fresh-frozen cadaveric knees (eight pairs) were affixed onto a customized knee-squatting simulator for measurement of femoral rollback and medial collateral ligament (MCL) strain during mid-flexion. One knee from each cadaver was randomly designated to the KA TKA group (with the joint line restored to the pre-arthritic condition) and the other to the MA TKA group . Optical markers were attached to all knees and rollback was analyzed using motion capture cameras. A video extensometer measured real-time variations in MCL strain. The kinematics and MCL strain prior to and following TKA were measured for all specimens. Results: KA TKA was better for restoring the knee kinematics to the native condition than MA TKA. The mid-flexion femoral rollback and axial rotation after KA TKA were consistently comparable to those of the native knee. Meanwhile, those of MA TKA were similar only at ≤40° of flexion. Furthermore, KA TKA better restored the mid-flexion MCL strain to that of the native knee than MA TKA. Over the entire mid-flexion range, the MCL strain of KA TKA and native knees were similar, while the strains of MA TKA knees were more than twice those of native knees at >20° of flexion. Conclusions: The restored joint line after KA TKA effectively reproduced the native mid-flexion rollback and MCL strain, whereas the altered joint line after MA TKA did not. Our findings may explain why patients who undergo KA TKA experience superior outcomes and more natural knee sensations during daily activities than those treated via MA TKA. Despite advances in technology and surgical technique, recent evidence indicates that mechanically aligned (MA) total knee arthroplasty (TKA) does not improve residual symptoms, natural knee sensations, or patient satisfaction ,2,3,4,5.Many daily activities, including walking and rising from a chair, are performed in the mid-flexion range ; restoraThe objective of this matched-pair study was to determine whether the restored pre-arthritic joint line configuration after KA TKA provided femoral rollback closer to that of the native knee than the altered joint line perpendicular to the mechanical axis created after MA TKA, and whether KA TKA more effectively restored MCL strain in comparison to MA TKA. We hypothesized that KA TKA reproduced the natural mid-flexion knee kinematics  better than MA TKA. In addition, we proposed that KA TKA would more naturally reproduce MCL strain in the mid-flexion range than MA TKA.Eight freshly frozen full-body specimens  were used . The twoThe specimens were frozen at 20 °C until they were thawed to room temperature on the evening prior to dissection. All skin and subcutaneous tissues were dissected away, leaving only the extensor mechanism, knee capsule, and periarticular soft tissues intact. We took high-resolution anterior-to-posterior photographs of each leg and measured the anatomical and mechanical axes of both the femur and tibia and the hip–knee–ankle axis. The severity of osteoarthritis (OA) in each cadaveric specimen was graded as mild (no articular cartilage lesions), moderate , or severe (extensive lesion present) . SeparatA senior surgeon (one of the authors) performed all arthroplasties following a standard posterior-substituting (PS) prosthetic system . Furthermore, a subvastus approach ensured exposure of the knee joint; the patella was not resurfaced in any case. We sought to ensure that all medial  resections were 9.5 mm in thickness, because the thicknesses of the distal and posterior femoral implants were 9.5 mm. KA TKA was performed using the previously described calipered technique ,28. The Following preparation, each knee was affixed in its original axial position onto a customized knee-squatting simulator system  ; this inA motion capture system combined with optical markers  was used to measure knee kinematics. Five motion capture cameras  were employed. Optical markers were attached to the medial and lateral epicondyles of the femur (the FML and FMM); medial and lateral ends of the longest medial-lateral axis of the tibia (the TBL and TBM); and RIG system  to analyze the medial and lateral femoral rollback B. We useReal-time variations in the mid-flexion MCL strain were examined with a noncontact video extensometer featuring a high-resolution digital camera  and real-time image processing software . The camera was placed 1 m from each knee . In order to minimize the effects of illumination, each test was completed under two 36-W light-emitting diodes. The MCL strain was assessed at knee flexion angles from 20 to 80° at intervals of 10°. The strain was measured over the entire MCL area; measurements were performed on each specimen prior to and following TKA B.t-test to determine whether the medial and lateral rollback, axial rotation, and MCL strain differed between the preoperative and post-TKA specimens. All analyses were completed using SPSS for Windows software , and a p-value < 0.05 indicated statistical significance. Additionally, an a priori power analysis based on a pilot test of changes in the femoral rollback and MCL strain of native knees was performed to determine the required sample size. We found that, for two-sided hypothesis testing at an alpha value of 0.05 and power of 90%, seven pairs of knees (14 knees) were needed to detect a 1-mm difference in the rollback and 5% difference in the MCL strain.All data were displayed as means with their corresponding standard deviations. We used the paired KA TKA restored the mid-flexion medial and lateral rollback and tibiofemoral axial rotation to levels closer to those of the native knee than MA TKA. The medial and lateral rollback of KA TKA and native knees was similar over the entire mid-flexion range A. The meKA TKA was better for restoring the MCL strain to that of the native knee over the entire mid-flexion range than MA TKA. The mean strain measurements following KA TKA and those of the native knee were alike over all ranges A. The MCDespite advancements in both technology and surgical techniques of MA TKA, patient dissatisfaction with post-TKA pain relief and overall outcomes remains high; a substantial proportion of patients report knee abnormalities ,5,30,31.The present study’s results endorse that KA TKA provides better physiological kinematics over the mid-flexion range than MA TKA, as hypothesized. We found that after KA TKA, medial and lateral femoral rollback, and axial rotation, were consistently similar to those of the native knee, whereas for MA TKA this was the case only at ≤40° of flexion. In addition, a paradoxical femoral forward movement during flexion was observed after MA TKA. Our findings agree with those of recent cadaveric studies, which suggest that compared to MA TKA, knee kinematics after KA TKA were more alike to those of native knees ,15,26. OOur findings also support the hypothesis that the joint line following KA TKA is better for restoring natural MCL strain during mid-flexion than the perpendicular joint line after MA TKA. KA TKA resulted in MCL strain that was consistently comparable to that of native knees, whereas, in the case of MA TKA, it was twice as high. Our findings agree with those of a recent cadaveric study: KA TKA was better for restoring the magnitude and distribution of MCL strain to natural levels than MA TKA . AlthougThe results of the present study propose that restoration of natural MCL strain via KA TKA may explain the more physiological knee kinematics evident after KA TKA compared to MA TKA. In this study, KA TKA reproduces more physiological medial pivot motion, while MA TKA results in paradoxical anterior motion during mid-flexion. Given that the MCL serves as the fundamental restraint in ACL-deficient, prosthetic knees, our findings suggest that MCL strain may be strongly associated with restoration of knee kinematics. Interestingly, a recent cadaveric study reported that restoration of joint line obliquity was not associated with mid-flexion coronal plane laxity, if the medial joint line height was restored . HoweverDue to the use of a cadaveric model, this study has some limitations. First, specimen preparation and the squatting loads used may not have been entirely natural. Second, as the tissue quality around the knee joint is associated with the severity of knee OA, the experimental results could be affected by the OA status of the cadaver. However, most knees in this study lacked advanced osteoarthritis necessitating TKA, so caution is required when extrapolating our findings to clinical practice. Third, this study featured PS prosthesis, which requires consideration of the implant feature prior to any broad generalizations, as implant design has been shown to be strongly correlated with knee kinematics ,35. GeneWe investigated whether restoration of the pre-arthritic joint line following TKA would affect post-TKA biomechanics. Restoration of the height and obliquity of the pre-arthritic joint line following KA TKA reproduces more natural rollback and MCL strain than alteration of the joint line following MA TKA over the entire mid-flexion range. Future studies focused on the development of both the motion analysis system that assesses the knee kinematics of patients in real clinical practice and the algorithm that recommends the optimal implant position restoring native knee kinematics are required.


Traumatic spinal cord injuries can have devastating outcomes for patients. In this focused review, we discuss the epidemiology of spinal cord injuries, associated neurologic exam findings, and primary and secondary injury progression. We then delve into the emerging treatment approaches and relevance to improving outcomes. The disease is multifactorial and has many management considerations. This concise user-friendly resource can help guide clinicians caring for these patients. Also, it points to the need for continued scientific discovery and improved pharmaceutical and device innovations. Traumatic spinal cord injuries (SCI) represent a serious and often irreversible neurological injury and can leave affected patients with catastrophic consequences to their quality of life. While there are several etiologies that can damage the spinal cord such as spinal tumors, demyelinating disorders, and infectious processes, trauma (particularly motor vehicle accidents) remains the most common mechanism of SCI in over 90% of cases. In 2020, there were nearly 18,000 newly reported cases of SCI1. In the USA alone, there are more than 300,000 people living with SCI [Initial workup often and at some point, during a patient’s hospital course includes computed tomography (CT) and/or magnetic resonance imaging (MRI) to assess severity and guide management decisions . MRI howThe ASIA impairment scale represents a standardized examination that incorporates motor, sensory, and anorectal examination with dermatomal consideration ,6. A tot7. Despite its prognostic ability, limitations exist with the ASIA scale as it does not assess anatomical implications of injury nor predicts other neurological sequalae of SCI. Furthermore, there is not enough evidence in the literature that differences in scores can or should be used to inform clinical decision making in regards to medical or surgical intervention [While the ASIA scale only assesses acute impairment, its prognostic value has been detailed in several studies –9. In a rvention The Frankel scale was previously utilized as a severity scale for SCI11. It was based on a 5-point scale (ranging from Grade A to E) that assesses motor function below the level of the lesion. Scores ranged from Grade A to E, with each score differing by varying levels of useful motor strength. In its conception, this provided a simple and standardized method for comparing SCI, but has since fallen out of favor with the development of the ASIA impairment scale.SCI can be classified into two phases of injury: primary and secondary. Primary injury results from the initial blunt force trauma to the spinal cord, affecting the neurons and glial cells at the location . Blunt sThe secondary phase of injury is triggered by the death of glial cells and ischemia at the site of trauma. Vascular permeability in the primary injury phase allows for the infiltration of immune cells to the site of inflammation . The chaThe main surgical intervention for spinal cord injury (SCI) involves spinal decompression, which aims to relieve the pressure on spinal microvascular circulation that often occurs from blunt spinal trauma. By relieving the pressure, the hope is to reduce hypoxia and ischemia , therebyIt is known that the level of the neurological insult correlates to the patient’s potential for recovery . There aIn regards to optimal timing of surgical intervention, the main thresholds currently reported in literature can be broken down into an ultra-early period (8–12 hours), an early period (within 24 hours), and a later period (48–72 hours) post-injury. It is important to note that these thresholds do not hold intrinsic biological relevance but are simply values chosen for practical implementation . ClinicaIn acute SCI medical management has become the mainstay of treatment alongside surgical intervention in improving patient outcomes. Medical management for acute SCI includes early augmentation of mean arterial blood pressure (MAP) , intensiAvoidance of hypotension and strictly adhering to blood pressure targets in patients with acute SCI serves to maintain adequate cerebral perfusion pressure and prevent secondary neuronal injury . OptimalWhile patients who suffer from acute SCI are hospitalized, they will also require intensive care management. Such care requires prophylaxis against deep vein thrombosis (DVT) and pulmonary embolisms within the first week after initial injury . The risUse of steroids was also once commonplace, but the benefits steroids have on decreasing inflammation are limited by severe systemic side effects . It is bAs alluded to above, searching for alternative treatment modalities that provide similar anti-inflammatory to steroids without its negative side effect profile necessitates further avenues of innovation. Several of these emerging modalities and compounds have been highlighted in recent literature, particularly in preclinical studies examining quality of life related outcomes following SCI. This body of data has continually increased over the past several years. There is heterogeneity in experimental intervention, but locomotor functional status and pain perception were the most common outcome measures. Therapies are aimed at mitigating both acute and chronic pathological processes, many targeting the complex inflammatory cascade that contributes to worsened paralysis and hyperalgesia. We describe some of these emerging compounds below and summarize our findings in –3.Betulinic Acid (BA) is a pentacyclic triterpene acid mainly extracted from birch bark. Recent studies using a rat model of cerebral ischemic MCA stroke demonstrated reduced oxidative stress and suppression of autophagy ,57. OxidPyroptosis is an inflammatory form of programmed cell death found to be decreased with BA administration based on inflammatory cascade markers. BA decreased levels of ASC, GSDMD, Capsase-1, NLRP3, IL-1B and IL-18 compared with the control group suggesting it plays a role in reduction of pyroptosis. To assess BA’s effect on autophagy LC3II, Beclin1, Vps34, CTSD, and p62 protein levels were assessed. These proteins describe autophagosomal markers. BA increased levels of all proteins indicating increased autophagy, the only substrate lowered was p62, an auto-phagocytic substrate protein (further suggesting autophagy up-regulation). Additionally, the AMPK-mTOR-TFEB activity was investigated. TFEB, known as the activator of autophagy, was increased alongside p-AMPK expression. This further suggests that BA activates autophagy and augments mitophagy. There is controversy regarding autophagy being beneficial or harmful in the context of SCI, but this paper found improved functional locomotor recovery in an animal model along with data to support the mechanism of action BA exhibits these effects .Historically, federal restrictions have limited research on cannabinoids (CBs) and related compounds in SCI. Aside from being the psychoactive constituent of marijuana, CB exist as endogenous compounds and modulation of these receptors may have therapeutic potential in dealing with chronic pain and affective disorders associated with SCI. Prior evidence exists supporting CB’s role in the CNS injury cascade. In a traumatic brain injury mouse model an endogenous cannabinoid, Arachidonoyl Glycerol (2-AG), may have a neuroprotective role. Levels were significantly elevated after brain injury and administration of synthetic 2-AG reduced brain edema and improved infarct volume and hippocampal death. 2-AG benefit was further evidenced by a dose dependent attenuation using SR-131761A, a CB1 receptor antagonist . This neA recent systemic review assessed the impact cannabinoid agonists  and antagonists (AM630) have on neurobehavioral outcomes in preclinical models of SCI. Animal models of injury included compression, contusion, ischemia-reperfusion, cryogenic, and transection . Agonists demonstrated statistically significant improvement on locomotor function in 9/10 studies and improve pain outcomes in 6/6 studies . CBs werPanax notoginseng used in traditional medicine acting as an antioxidant. There are several extracellular molecules that inhibit synaptic sprouting and neuronal plasticity. Nogo-A appears to be the most important of these inhibitory ligands and this soluble decoy receptor binds to Nogo-A, encouraging anatomical growth and regeneration of neurons. NgR1 has been demonstrated in prior studies to alleviate glutamate induced oxidative stress, apoptosis, and mitochondrial dysfunction via SIRT1 activation of the WNT/B-Catenin pathways [Notoginsenoside Receptor 1 (NgR1) is an active compound isolated from pathways ,61.61. The SCI model used was a C5/C6 right hemi section followed by one treatment arm receiving chronic intrathecal and IV NgR1-Fc every other day. Histological analysis demonstrated a 250% increase in corticospinal axon density in the cervical cord below the level of the injury when compared with control. Behavioral analysis showed increase in affected right arm use from 6% in the control to 17% in the experimental group.A recent study done on cynomolgus monkeys and rats investigated a soluble decoy receptor for a myelin-associated inhibitor NgR1. The soluble decoy NgR1-Fc AXER-204 has been promoted recovery in a series of preclinical rodent models, but this most recent study proved safety and efficacy in a non-human primate. Results showed no toxicology or safety concernsRiluzole is a glutamatergic neurotransmission inhibitor with survival benefit in amyotrophic lateral sclerosis (ALS). Riluzole’s mechanisms of action is not completely understood. It is theorized the impact is secondary to a multifactorial process altering synaptic concentrations of excitatory amino acids. It is proposed to do so by blockage of sodium channels, antagonizing both NMDA and non-NMDA receptors, and GABA reuptake inhibition . This beThere is a body of evidence demonstrating neuroprotective, anti-ischemic, and anti-epileptic properties of riluzole  . In a rAnother study compared riluzole head-to-head with two other treatment modalities showing preclinical promise, hypothermia and glibenclamide. These three treatments were specifically identified to interfere with progressive hemorrhagic necrosis (PHN), which is irreversible conversion of healthy tissue secondary to advancing microvascular dysfunction. With existing preclinical data coming from a variety of labs using differing SCI injury models and outcome assessment, this study provided valuable direct therapeutic comparison under a controlled environment. Compounds were delivered four hours after trauma using a unilateral impact injury model at the C7 cord producing isolated ipsilateral hemorrhage. Riluzole was dosed at 8 mg/kg IP twice daily, glibenclamide was dosed 10 µg/kg IP loading dose followed by a one-week long subcutaneous pump delivery of 400 ng/hour, and systemic moderate hypothermia  was induced for four hours under anesthesia.Direct comparison revealed favorable results in all three compounds compared to control. The hypothermia and glibenclamide treatments performed in a similar manor, both outperforming riluzole treated rats in the first two weeks. Prior to day seven riluzole also showed a higher degree of autonomic dysfunction compared with other treatments . After six weeks results between the compounds were identical and lesion volume assessed at the time was smaller in all three groups. Largest reduction in lesion volume was noted in the glibenclamide treatment followed by hypothermia and riluzole, respectively. After injury all groups showed atrophy of the spinal groups rostral to the lesion except riluzole. Of the three, riluzole showed higher mortality rates attributed to its unfavorable therapeutic index. Final comparison results concluded that all are strong candidates for translation into clinical trials and that systemic hypothermia and glibenclamide may have a greater efficacy and safety profile compared to riluzole .Repulsive guidance molecule A (RGMa) is a molecule important for the processes of cell migration, differentiation, and apoptosis of different organs . SeveralRecent studies have explored Elezanumab impact on functional neurological recovery in the clinical and preclinical context. The antibody is currently being investigated in Phase II clinical trials for multiple sclerosis, acute spinal cord injury, and acute ischemic stroke. In a mice model of noise induced cochlear damage, RGMa antibody improved synaptic regeneration .A recent series of five studies was conducted by Jacobson  and collTNF-a is known to be one of the most potent pro-inflammatory cytokines and is elevated following acute SCI ,70. LargThe data obtained on sTNFaR1 used a C5 unilateral cervical contusion model. Immediately following the contusion, 10 µL of sTNFaR1 was delivered intrathecally over 5 minutes. A series of new experiments revealed that a 90-minute delayed bolus dose to the contusion reduced expression of neuroinflammatory markers and consistently improved neurological function over a six-week period . This stDelayed administration of high dose human immunoglobulin G (hIgG) was investigated by Chio et al. using an animal SCI model on Wistar rats . Prior sDelayed administration of high dose human immunoglobulin G (hIgG) was investigated by Chio et al. using an animal SCI model on Wistar rats . Prior sTraumatic spinal cord injury represents a significant neurological insult with high morbidity and mortality. It is associated with devastating acute and long-term neurological outcomes with limited avenues for meaningful intervention. SCI often occurs as a result of blunt force trauma and a resulting inflammatory cascade due to secondarily compromised vascular supply, pro-inflammatory mediators, and neuronal dysfunction due to changes in the microenvironment. In the acute phase of injury, neurological impairment should be assessed as quickly as is reasonably possible. Clinical tools such as the AIS scale can assess neurological impairment and provide some prognostic information. Although its prognostic ability has been validated in several studies, it has not yet been proven useful in informing immediate clinical decisions regarding medical and surgical intervention.Outside of medical management involving adequate hydration and hemodynamic support, surgical decompression has been the mainstay of treatment to relieve pressure from the spinal cord as a result of edema or other surrounding tissue damage. The notion of “time is spine” has been a predominant theme, with studies showing benefit in surgical intervention within 24 hours of injury. However, there remains substantial contention in regards to the optimal timeframe for surgical intervention. Current and future studies will be required to fully understand the best timing for surgical intervention and help stratify patients that stand to benefit from these invasive procedures.Despite appropriate surgical and medical intervention patient’s often do not regain meaningful neurological function following SCI. Emerging treatment modalities have offered some hope however in pre-clinical studies. Organic compounds, small molecules, and antibodies have shown considerable promise in modifying the SCI cellular microenvironment in favor of more positive outcomes. These experiments have mainly been carried out in murine and non-human primate models with varying success. More research will need to be conducted to better assess the efficacy and safety profile of these emerging treatments with the hope of advancing more of these novel interventions to human clinical trials.


To conduct a systematic review of the effectiveness of facial exercisetherapy for facial palsy patients, updating an earlier broader Cochranereview; and to provide evidence to inform the development oftelerehabilitation for these patients.MEDLINE, EMBASE, CINAHL, Cochrane Library, PEDro and AMED for relevantstudies published between 01 January 2011 and 30 September 2020.Predetermined inclusion/exclusion criteria were utilised to shortlistabstracts. Two reviewers independently appraised articles, systematicallyextracted data and assessed the quality of individual studies and reviews. Thematic analysis used forevidence synthesis; no quantitative meta-analysis conducted. The review wasregistered with PROSPERO (CRD42017073067).n  =  854 participants). 75% utilised validated measuresto record changes in facial function and/or patient-rated outcomes.High-quality trials (4/7) all reported positive impacts; as didobservational studies rated as high/moderate quality (3/9). The benefit oftherapy at different time points post-onset and for cases of varyingclinical severity is discussed. Differences in study design prevented datapooling to strengthen estimates of therapy effects. Six new review articlesidentified were all rated critically low quality.Seven new randomised controlled trials, nine observational studies, and threequasi-experimental or pilot studies were identified(The findings of this targeted review reinforce those of the earlier moregeneral Cochrane review. New research studies strengthen previousconclusions about the benefits of facial exercise therapy early in recoveryand add to evidence of the value in chronic cases. Further standardisationof study design/outcome measures and evaluation of cost-effectiveness arerecommended. The cause still unclear, although the condition has long been associated with reactivation of latentvirus infection, with evidence showing a rise in incidence in the United States thought to belinked to increasing herpes infection rates. The majority (60%) of these facial nerve paralysis cases are Bell's palsy, and this condition affects 11–40 people per 100,000 in the population eachyear, most commonly in the age group 30–45. It is also estimated that one in sixty individuals will be affected over thecourse of their lifetime.8Population studies show that facial palsy is more commonly associated with peoplewho are immunocompromised or pregnant, or those with obesity, hypertension,diabetes, or upper respiratory conditions.9 With the appearance of COVID-19caused by SARS-CoV-2, reports are also emerging of Bell's palsy as a presentingsymptom this for infection.11 Although most patients eventually recover without anytreatment,12 one in three (29%) have a poor recovery resulting in apermanent deficit of facial function.13 This can affect importantfunctions such as eating and speaking, or non-verbal communication mediated by expression of emotion (e.g. smiling), as well as chronic facial pain.16 Patients living withincomplete recovery experience long-term psychological distress and depression, withmany moving away from public-facing roles resulting in social alienation.1619Bell's palsy is the most common form of acute spontaneous peripheral facialparalysis, with poor recovery affecting a patient's long-term quality of life. These range from medication and physical therapies to surgery. Evidence ofthe effectiveness of surgical interventions remains limited, with a Cochranesystematic review reporting insufficient proof of benefit and further trials judgedto be unlikely. Cochrane reviews have identified one pharmaceutical treatment (prednisolone)as an effective treatment (versus placebo) for Bell's palsy, but only ifadministered within 72 h of symptom onset.2224 Use of adjunctive antiviraltherapy was reported to be of uncertain value, although more recent evidence of its value in the treatment of Ramsay Huntsyndrome, the second most common cause of facial palsy, has emerged. In addition to medication, a number of physical therapy options areavailable. Facial exercise therapy  is the mostwidely evaluated.2631 A Cochrane review of physicaltherapies published in 2011 reported some evidence that facial exercise therapycould improve facial function for moderate paralysis and chronic cases and reducesequelae in acute cases, but it recommended the need for further studies. We have therefore undertaken a systematic review to identify and appraise anyfurther studies that have examined the effectiveness of facial exercise therapy.Updated evidence on effectiveness is required to inform the planned development offacial remote activity monitoring eyewear to enable telerehabilitation in apatient's own home.33The evidence base for therapy in acute and chronic cases remains limited. Currently,a range of treatment options may be offered, with a recent study identifyingconsiderable variation in the care pathways experienced by patients in the United Kingdom. A mixed-methods approach to evidence synthesis was specified in the protocolto allow for the inclusion of different study designs, in addition to randomisedcontrolled trials.36 The peer-reviewed protocol also specified the inclusion ofevidence from post-2011 reviews to maximise the evidence identified. There wasstrict adherence to international guidelines on Preferred Reporting Items forSystematic Reviews and Meta-Analyses (PRISMA).The review aimed to identify and assess recent evidence of the effectiveness offacial exercise therapy, draw out any clinically relevant findings, and identifyfuture research needs. The review protocol was submitted for peer-review to theInternational Prospective Register of Systematic Reviews (PROSPERO), ID: CRD42017073067. Tailored search strategies were used for the six databases and details arepresented in Appendices 1–6.Literature searches were undertaken by an Academic Librarian (CB) qualified insystematic reviews. Databases searched included MEDLINE, EMBASE, CINAHL, CochraneLibrary and PEDro; plus the Allied and Complementary Medicine Database). The latter was notincluded in the original Cochrane review. Searches were replicated covering theperiod 1 January 2011 to 30 September 2020, allowing for a slight overlap with thesearch end date (28 February 2011) of the earlier Cochrane review.Abstracts were screened against predetermined inclusion/exclusion criteria see , and absShortlisted articles were read carefully and assessed by two independent readers (AKand DT) for inclusion in the review. Some articles were discarded if these repeatedinformation provided in another version. Details of all articles were entered intoan Endnote library and bibliographies were scrutinised to identify possibleadditional studies. Screening and shortlisting data were presented as a PRISMA flow diagram.For data extraction and management, articles were organised into two groups (i)individual primary research studies and (ii) reviews. Summary Tables were pilotedfor data extraction from both types of articles. Common information extracted fromall articles included author names, publication date, and country of publication.For individual research studies, additional data recorded included: study design;sample size & patient demographics; details of clinical diagnoses/conditions;details of intervention and comparator(s); outcome measures; main findings; andconclusions. For review articles, data extracted also included type and number ofstudies reviewed, publication dates, and total number of patients. Information wasextracted and entered into the Summary Table initially by two author . Thiswas then checked and amended and expanded where necessary by other reviewers,separately for individual studies  and for reviews .39 Assessmentwas performed independently by pairs of reviewers . Studies werecategorized into three levels of bias ; a thirdresearcher (AS) assisted with any disagreements that arose. For the publishedreviews shortlisted, quality was assessed using AMSTAR-2 by two independentreviewers . Confidence in the quality of each review was categorised ashigh, moderate, low, or critically low, based on the number of critical andnon-critical flaws identified.Quality assessment was undertaken using validated tools. For individual researchstudies, quality and risk of bias were assessed using GRADE  appraisal tools. Where available, the results of individual randomised controlled trials werereported separately, as were the findings of cohort studies. The authors coded allthe studies before discussing and agreeing on descriptive categories and themes forsynthesis.In preparation for final analysis, all papers were read in full, with data andresults extracted, regardless of the study design . For individual studies, data entered into the Summary Tableincluded study design, diagnoses, therapies considered, results, limitations, andstudy quality rating. A further table collated the main findings and conclusionsdrawn . As anticipated, authors used differing study designs,papers considered a mix of therapies and controls, and adopted different outcomemeasurement tools. Statistical combination of numerical data from separate studiesto strengthen review conclusions  was not possible due to variationsin study design and quality. A thematic analysis framework was therefore used tocreate a descriptive synthesis and address the heterogeneity of studies identified. Brazil, Iran, Italy, New Zealand, and Portugal.Characteristics of included studies: The 19 individual studies shownin n = 16 patients); two pilot studies (n = 37 patients)50;and ninenon-experimental observational studies (n = 447 patients).Observational studies varied in their design: three were cohort studies(n = 169 patients)5153; two were retrospectivestudies (n = 101 patients)55; three used group comparisons(n = 164 patients)5658; and one a small clinicalstudy of 13 patients. In total, the research studies identified reported the impact of physicaltherapy in a total 854 participants (age range 12–80 years old).Methodological quality and risk of bias: Consensus ratings of themethodological quality of studies (GRADE) are shown in the final column of 6063; and one was a cohort study. A further six studies were judged to be of moderate quality. Two wererandomised controlled trials,65 and four werenon-experimental observational studies.58 The remaining nine studieswere rated as low methodological quality. Two were randomised trials,66 one was aquasi-experimental study, and two were small pilot studies.50 In terms of methodologicalflaws all, except one study, were at risk of bias for failing to blind participants when outcomes werebased on patient-reported measures. One high-quality study failed to achieve therequired sample size, with a significant number of severe facial palsy patients inthe control group dropping out in order to access physical therapy which was shownto be improving outcomes.Patient characteristics (diagnoses/conditions): There was a highlevel of heterogeneity in the characteristics of patients included in the studies asshown in  five days, ten days, to one month since onset.51 One study recruited patientsin the ‘sub-acute phase’, that is, 3–5 days after the acute onset subsided. A further two studies focused on chronic stages of recovery, that is, 2–30years since onset.64 The remaining 11 studies did not pre-specify time post-onset atstudy entry. Individual studies also varied in the types of facial palsy patientrecruited. All studies included Bell's palsy patients. Three studies also recruitedsome Ramsay Hunt syndrome patients.66 Where specified, all patientshad unilateral facial palsy, although some studies did not state the type. Somestudies recorded patients’ level of facial synkinesis at entry.59 Othersfocused on the severity of facial palsy; this ranged from Grade II to VII.64Therapies Evaluated: Only two studies evaluated the use of facialmuscle strengthening exercises on their own.55 A further five studiesevaluated facial exercise therapy combined with biofeedback (via mirror or otherdevice)5154; and one evaluated exercisewith added facial taping. One paper compared Western facial exercise therapy with or without theaddition of a Korean variant of facial muscle exercises. The remaining studies reported the effects of combining facial exercisetherapy with another form of treatment. Two studies explored the use of lasertreatment before or in conjunction with facial exercises,65 and two research teamsassessed the addition of electrical stimulation to physical therapy.63 Three papersexamined physical therapy with and without corticosteroids62; and three studies consideredBotox added to facial therapy.64Outcome measures reported: Individual studies used three mainvalidated measurement tools (see n  =  9 studies). A further six studiesincorporated the Sunnybrook Facial Grading Scale (SB-FGS) to measure the severity offacial paralysis symptoms. A patient-rated outcome measure of the disabilityresulting from facial palsy, the Facial Disability Index (FDI), was used in sixstudies; one study reported separate results for the physical elements (FDIP) andsocial elements (FDIS) of the FDI. The five remaining studies utilised a different single measure; two used analternative validated facial grading system,67 and three used anotherunvalidated measure.66 All these five studies were assessed to be of low quality.Outcome measures used and their timing varied in the four high-quality randomisedcontrolled trials,6063 meaning comparable data werenot available for meta-analysis to be undertaken. The choice of outcome measure(s) was not indicative of the final quality(GRADE rating) of studies, although highly rated studies tended to use a combinationof measures. For the randomised controlled trials, domains in which GRADE identifiedcritical flaws are identified in ools see . Further randomised patients with HBS scores of 2–4 into electrical stimulationwith exercise or exercise-only treatment groups. They reported a statisticallysignificant improvement in HBS scores at three months after onset in thecombined treatment group but no difference in FDI scores between the groups. Theexclusion of HBS grades 5 and 6 and the limited sensitivity of the HBS scalemakes it difficult to draw any conclusions for these patients. Alayat et al. found that high-intensity laser therapy, massage, and exercise threetimes a week for six weeks provided statistically significant improvements inHBS and FDI scores in those in the acute phase of unilateral Bell's Palsy whencompared with low-intensity light therapy with massage and exercise or massageand exercise alone; the exercise-only group showed the lowest effect. Nicastriet al. reported physical therapy produced a significant improvement in HBSgrade, but time to recovery only improved in those with severe (HBS 5/6) facialpalsy. Ferreira et al. reported, with the exception of resting symmetry of the cheek and mouthon the SFGS, that neuromuscular retraining was as effective as acute provisionof corticosteroids in improving static and dynamic symmetry in those withunilateral idiopathic facial palsy. The final, observational study, DallaToffola et al. found no difference in the recovery in patients with idiopathic facialpalsy and evidence of axonometric facial nerve injury when provided with EMGbiofeedback compared to mirror biofeedback as a primary rehabilitation tool.Development of synkinesis, extent of motor recovery, and health resources usedwas included in their appraisal of recovery.Among the five high-quality articles, four were randomised controlled studies allof which reported a positive effect. Tuncay et al. completed a small trial of 20 consecutive patients randomly allocated topre-treatment with Botulinum toxin type-A prior to standard facial therapy. Theyfound that both groups showed improved outcomes on the SunnyBrook scale, butpre-treated patients had significantly better outcomes. Ordahan and Karahan randomized 46 patients with acute idiopathic palsy to physical exercisewith or without low-level laser therapy. They reported that therapy alone showeda significant improvement in FDI score from baseline at week 6 but not at week3. Laser therapy and exercise produced significant improvements from baselineFDI scores at both three weeks and six weeks post onset.The six articles rated as medium quality included two randomised controlledtrials and four non-experimental observational studies. All reportedimprovements Monini et al. reported a statistically significant increase in SFGS, FDIP and FDIS whenmirror therapy was combined with standard facial rehabilitation. Standardrehabilitation was documented as including massage, stretching, neuromuscularre-education, myofascial release, and postural exercises. Monini et al. found that treating all age groups with steroid therapy and physicalrehabilitation resulted in a significantly quicker time to recovery than steroidtherapy alone. They also highlighted the importance of physical therapy forolder patients and those with more severe HBS grading at the onset of theirpalsy. This followed their previous trial showing standard facial therapyresults were improved with the addition of preventive Botulinum toxin treatment. Kim et al. compared Western physical therapy with Korean symmetric self-performedfacial muscle exercises, reporting that the addition of SSFE showed significantimprovement in HBS at four weeks. In a study by Fujiwara et al., physical rehabilitation was shown to prevent significant deterioration insynkinesis in female and younger patients with Bell's palsy.Among the four observational studies rated as medium quality, Barth et al.Pvalues). One reported a non-significant difference between two methods ofbiofeedback , while all others demonstrated a significant improvement.6165 No studies attempted tocalculate effect size, so there were insufficient data to assess imprecision orinconsistency as outlined in GRADE approach.Eight of the 11 individual studies assessed to be of medium to high qualityincluded the statistical significance of their results  reported no significant effect, and two studies simply reported percentage improvements.59 In theone poor quality randomised controlled trial, Di Stadio et al. reported a significant improvement for Kabat rehabilitation with andwithout facial taping based on a new rating Arianna Disease Scale.Among the remaining nine individual research studies that were assessed to be oflow quality, three articles reported a statistically significant effect rangingfrom  compared the efficacy of EMG and mirror biofeedback over a 12-monthperiod. Ferreira et al. assessed the role of corticosteroids (prednisolone) and neuromuscularretraining in recovery from acute idiopathic palsy and so chose to perform theiranalyses at the entry to the study and six weeks later. Kim et al. reviewed acute outcomes at four weeks post onset. Fujiwara et al. appraising physical therapy in the treatment of synkinesis selected 6, 9and 12-month intervals to assess the outcomes. Nicastri et al. followed study participants up on a monthly basis for six months.The timescales over which physical therapy was assessed differed widely and thislimits any robust analysis of how sustainable the reported therapeutic effectswere. Dalla Toffola et al. The six new reviews were published over the period 2011–2017 andoriginated from research groups in Australia, Brazil, Iran, Italy, New Zealand, and Portugal. Apart from the 2011 Cochrane review by Teixeira et al., all systematicreviews were rated as ‘critically low’ using the AMSTAR-2 rating. This found low-quality evidence that tailored facial exercise couldreduce sequelae in the acute phase, could improve facial function in those withmoderate paralysis, and could produce improvements in the chronic stages. Theauthors concluded that further research evidence was required, as provided byindividual studies listed in 4247 But all have majormethodological shortcomings resulting in critically low AMSTAR-2 qualityratings, so could not be included in our synthesis. Critical weaknesses includelack of clarity a priori about the review protocol; deviationsfrom the original protocol; a lack of comprehensive description of searchstrategies, excluded studies, or risk of bias assessments; and no considerationof how bias might influence the interpretation of results. Equally important,the reviews did not include additional new evidence; only half identified anystudies published since the 2011 Teixeira review. Pourmomeny and Asadi identified two new randomised controlled trials; Ferreira et al. included one new study; and the 2017 review by Fargher and Coulson identified two randomised controlled trials. Only three of these fivestudies met our inclusion criteria and they had already been included in ourreview63; the other two did not focus on facial exercisetherapy.71The six more recent reviews all reach largely positive conclusions about theeffectiveness of facial exercise therapy. focusing on tailored facial exercise therapy. The review identified nineteennew research studies, including eight randomised controlled trials, fourquasi-experimental or pilot studies, and seven observational studies. These studiesprovide evidence on the effectiveness of facial exercise therapy in 839 patientswith ages ranging from 12 to 80 years old. Although, six reviews published after theCochrane review were also identified all were rated as being of critically lowquality. They also identified only three new studies which met our inclusioncriteria, and all these were already included in our review. This article thereforeadds significantly to previously available evidence on the effectiveness of facialexercise therapy for the recovery of people diagnosed with facial palsy.The primary aim of this study was to update the evidence base on the effectiveness ofphysical therapy for facial palsy rehabilitation,58The 19 new studies included in this review vary in their research design. Sevenincorporated a randomised controlled trial, nine used a non-experimentalobservational design, and the remainder were quasi-experimental or pilot studies.Overall, four out of seven trials were rated as high quality and all described apositive impact. Among the nine observational studies, three were rated as high ormoderate quality, all cohort studies. Two of these studies reported a significantlybetter outcome for those receiving facial exercise therapy, especially among youngerpatients.55 Six studies assessed physicaltherapy combined with biofeedback, either via a mirror or other means; all reportedpositive improvements.5166The effectiveness of facial exercise therapy on its own was evaluated in only twostudies; both reported that patients performing facial exercises achieved greaterfunctional recovery than those who did not.62 botulinum toxin,64 electricalstimulation,63 or laser treatment.65 Nine reported a positiveimprovement.The remaining 10 studies evaluated the effectiveness of combining facial exercisetherapy with corticosteroids,66 A further 12 studies focusedon chronic cases including patients from 2 to 30 years post onset.64 These studiesstrengthen previous Cochrane review evidence on the value of physical therapy earlyin recovery, and also add to the previously limited evidence in support of its usefor chronic cases. Evidence of the specific benefits with increased clinical severity is lessclear. Five research studies selected patients based on the severity of their facialpalsy, with cut-offs ranging from Grade II to VII,64 and a further three reportedselecting patients based on the presence of facial synkinesis.59 However, itis not possible to draw any conclusions on the impact of physical therapy dependingon the clinical severity of facial palsy from these studies.This review considerably strengthens the evidence base in support of facial exercisetherapy, but information on the specific benefits of therapy at different timepointspost-onset of facial palsy or for patients living with different levels of severityis difficult to disentangle with certainty. In studies where the stage of recoverywas specified, six studies concentrated on the acute phase, recruiting patients from48 h to one month since onset of facial paralysis. A clinician facial grading system (House–Brackmann or Sunnybrook) was used in12 studies, and a patient-rated outcome measure  wasincluded in six studies. The use of both a clinician and patient outcome measure wasobserved in the highly rated studies. A recent systematic review has recommended theadoption of Sunnybrook for reporting outcomes of facial nerve disorders. Only three studies included in the review used a single, non-validatedoutcome measure; all were rated low quality. No study defined exante what would represent a clinically significant change. A similarfinding has been reported in the service setting when monitoring patient recovery. No study included cost-effectiveness as an outcome or incorporated ahealth-utility outcome measure (e.g. EQ-5D) that would enable economic modelling. Finally, because the point at which outcomes were measured ranged from 4weeks to 12 months post-treatment, evidence on the sustainability of anyimprovements following therapy is difficult to gauge. Arguably, a comprehensiveanalysis of the impact of therapy on the evolution of facial palsy, includingsynkinesis, should be performed over a minimum of a 24-month period.The review findings also demonstrate an increase in the use of validated outcomemeasures, compared to research identified in the earlier Cochrane review. The heterogeneous nature of the current evidence base also limits the abilityto draw any clear correlation between the effectiveness of facial exercise therapyand time since onset of facial palsy, clinical severity, or other patientdemographics. The use of both a clinician grading system and patient-rated outcomemeasure, as identified in high-quality studies, is recommended.This review provides new evidence from high-quality studies to support the use offacial exercise therapy in patients with facial palsy. Limitations of the evidenceinclude differences in research design, the interventions evaluated, and patientsincluded in studies. This heterogeneity makes it impossible to pool data fromindividual studies to increase the power and precision of estimates of treatment effects. as well as possible links to vaccines,77 with safety concerns alreadyofficially recorded for the latter, and a recent call for surveillance following use of particular vaccines. Large-scale COVID-19 vaccine programmes could lead to a rise internationallyin the future number of facial palsy cases. Our review demonstrates widespreadinternational interest in evaluation of physical therapy for facial palsy, withstudies originating from 10 different countries. This provides a good foundation forfuture collaborative research, moving away from a previous pattern of studies almostexclusively undertaken in the United States. International collaborations are already leading to standardisation ofoutcome measures, and development of value-based reimbursement strategies. A future move to digitally enabled facial palsy rehabilitation at home isalso likely to be catalysed.34 Pre-pandemic, a review oftelerehabilitation could identify no facial palsy studies. This situation is likely to change as a consequence of an acceleratedworldwide move to digitally enable services due to COVID-19, new routes for commissioning home-based digital services,84 and thedevelopment of new wearable technologies.Looking to the future, further improvements to the evidence-base will become evenmore important if reports emerging of a link to COVID-19 are substantiated. Theseinclude a tripling of cases in patients who have had COVID-19, Because this form of central facial palsy is clinically distinct, current review findings are not directly transferrable. But, the lessonslearned here might be applied to improve the evidence base for these patients.This review has concentrated on peripheral facial nerve paralysis. A similar evidencebase for facial palsy following stroke is currently lacking.RCTs of facial exercise therapy confirm improved facial functioning andpatient-reported outcomes.Therapy is effective in early cases, when combined with other treatments,and potentially in chronic cases.Multi-centre collaboration is needed, particularly given emergingevidence of links with COVID vaccination.Specialist facial therapy should be integrated fully into treatmentpathways.


Mycobacterium chimaera infection is an emerging disease in people undergone to cardiothoracic surgery, which need to be suspected also with atypical presentations.Disseminated M. chimaera and a specific antimicrobial therapy was started, with apyrexia after 7 weeks.We report the case of a 74-year-old man with fever of unknown origin, purple nodules on both feet and a history of open-heart surgery. Imaging investigations showed an abscess near aortic bioprosthesis but screening for endocarditis resulted negative and pyrexia did not respond to antibiotic therapy. A biopsy of cutaneous lesions showed HHV8-related Kaposi’s sarcoma, so bone marrow biopsy was executed with evidence of HHV8 localization. Bone marrow and urine mycobacterial cultures resulted positive for M. chimaera infection should be always investigated as a possible etiology of fever of unknow origin in people with a history of open-heart surgical intervention, even with negative mycobacterial blood cultures. The possible role of disseminated infection in inducing immunodepression with the occurrence of other opportunistic diseases (such as Kaposi’s sarcoma) cannot be excluded. Mycobacterium chimaera is an opportunistic, water-borne, non-tuberculous mycobacterium (NTM), belonging to the Mycobacterium avium complex, first described in 2004  were negative. We also performed screening for non-infective endocarditis , with a negative result. Because of the persistence of fever despite multiple antibiotic treatments , stool (BD BACTEC Mycobacteria Growth Indicator Tube 960 system and Lowenstein-Jensen medium) and bone marrow (BacT/ALERT®3D automated culture system) cultures were executed, with isolation of M. chimaera from urine and bone marrow , a 70-year-old man underwent coronary artery bypass graft and aortic valve replacement with bioprosthesis, needing extracorporeal circulation. His past medical history included hypertension and chronic kidney disease. In June 2021 he was hospitalized in our Infectious Diseases Unit for pyrexia from more than one month  were first described in an Italian immunocompetent HIV-negative woman with HHV8 infection, who developed KS after 10 months from the appearance of fever . After 5M. chimaera infection contributed to the occurrence of KS is unknown. Actually, mycobacterial bone marrow involvement might have had a role in the development of pancytopenia and reduced bone marrow and peripheral blood lymphocyte count with inverted CD4+/CD8+ ratio, which in turn could have favored the appearance of HHV8-related manifestations, similarly to what had been described in people living with HIV and in individuals with organ or bone marrow transplantation [As what concerns the pathogenesis of KS in our patient, Classic Mediterranean KS occurs typically in middle-aged and elderly HIV-negative people with HHV8 infection and is characterized by purple papules or nodules predominating in the lower limbs . In thisantation .M. chimaera infection in people with fever of unknown origin and previous cardiothoracic surgical intervention with need of extracorporeal circulation, even when initial mycobacterial blood cultures and echocardiographic investigations are negative. The absence of other cases reported in the same Institute cannot let the clinicians reject this diagnosis. Disseminated mycobacterial infection with bone marrow involvement might also explain, partially at least, a condition of immunodepression and therefore coexist with opportunistic diseases, as KS.In conclusion, the case reported underlines the necessity of a complex diagnostic workflow to exclude


Liposomes and planar membranes made of archaea or archaea-like lipids exhibit many unusual physical properties compared to model membranes composed of conventional diester lipids. Here, we review several recent findings in this research area, which include (1) thermosensitive archaeosomes with the capability to drastically change the membrane surface charge, (2) MthK channel’s capability to insert into tightly packed tetraether black lipid membranes and exhibit channel activity with surprisingly high calcium sensitivity, and (3) the intercalation of apolar squalane into the midplane space of diether bilayers to impede proton permeation. We also review the usage of tetraether archaeosomes as nanocarriers of therapeutics and vaccine adjuvants, as well as the biomedical applications of planar archaea lipid membranes. The discussion on archaeosomal therapeutics is focused on partially purified tetraether lipid fractions such as the polar lipid fraction E (PLFE) and glyceryl caldityl tetraether (GCTE), which are the main components of PLFE with the sugar and phosphate removed. Archaea tetraether lipids are, in most cases, bipolar macrocyclic compounds containing dibiphytanyl (2 C40) chains. Archaea diether lipids also adopt the sn-glycerol-1-phosphate stereochemistry and usually contain diphytanyl (2 C20) chains. Many archaeal lipid analogues have been chemically synthesized, with their physico-chemical properties characterized  = 0.1–1.0 mM at pH 7.6 [PLFE has been explained in terms of strong PLFE-PLFE (thus tight membrane packing) and MthK-PLFE interactions [PLFE is most likely to be significantly different from that in BLMdiester. It was proposed (a (BChla) in the light-harvesting polypeptide (LH)/BChla complex, which was found to be more homogeneously and perpendicularly oriented with respect to the membrane surface in archaea tetraether lipid membranes than in diester lipid membranes [2+ to the MthK RCK domains, which triggers a series of protein conformational changes, leading to channel opening and K+ conduction  may render this statement incorrect. In these planar membranes, fluidity is maintained by a thin layer of trapped water (2–10 Å) between the substrate and the bilayer, allowing for more surface-specific analytical techniques to be utilized [Phospholipid bilayers supported by a solid substrate are thought to be more stable and robust compared to black lipid membranes made of conventional diester lipids. Recent progress on BTLutilized .To date, the applications of solid-supported planar membranes made of archaea or archaea-like lipids are largely limited to biophysical and electrophysiology studies. The physical tools used for these studies include Langmuir-Blodgett films, atomic force microscopy, tensiometry, ellipsometry, small angle X-ray scattering and fluorescence microscopy. The major findings include (1) the stereochemistry of a central cyclopentane ring is critical for controlling the conformation of the corresponding bipolar tetraether lipids ,125; (2)Thermoplasma acidophilum and was used on the surface of cellulose dialysis membranes [T. acidophilum tetraether lipids, on the other hand, were removed to yield the bare hydrophobic core, which were then reacted with cyanuric chloride at both polar headgroups. When cyanuric chloride-activated caldarchaeols in aqueous solution are spread to the solid support [Due to the high stability against various environmental stressors and the bipolar nature of the chemical structure, archaeal tetraether lipids are attractive coating materials to modify the surfaces of medical devices and implants to prevent or reduce biofilm formation and bacterial infections. An immobilization matrix was developed from monolayers formed by bipolar tetraether lipids isolated from the archaeon embranes  and mediembranes . Specifiembranes ) on T. a support , the cya support . Such ar support . The biv support . HoweverPLFE (Despite the fact that many efforts have been devoted to studying archaea lipid membranes and finding their applications in the past four decades, the unusual and interesting physical properties of membranes containing archaea or archaea-like lipids have been reported in recent years. In this article, we have particularly reviewed the works of apolar squalanes in diether bilayers , thermosPLFE . These f


We use phylogenetics to show that Zam proteins are distributed across cyanobacteria and that they form their own unique clade of the ribonuclease II/R (RNB) family. Despite being RNB family members, multiple sequence alignments reveal that Zam proteins lack conservation and exhibit extreme degeneracy in the canonical active site—raising questions about their cellular function(s). Several known phenotypes arise from the deletion of zam, including drug resistance, slower growth, and altered pigmentation. Using room-temperature and low-temperature fluorescence and absorption spectroscopy, we show that deletion of zam results in decreased phycocyanin synthesis rates, altered PSI:PSII ratios, and an increase in coupling between the phycobilisome and PSII. Conserved cysteines within Zam are identified and assayed for function using in vitro and in vivo methods. We show that these cysteines are essential for Zam function, with mutation of either residue to serine causing phenotypes identical to the deletion of Zam. Redox regulation of Zam activity based on the reversible oxidation-reduction of a disulfide bond involving these cysteine residues could provide a mechanism to integrate the ‘central dogma’ with photosynthesis in cyanobacteria.The  Cyanobacteria are photosynthetic prokaryotes that play major roles in the biosphere—serving as the major fixers of nitrogen in the ocean and as primary producers . As theizam) gene was first identified in the cyanobacteria Synechocystis sp. PCC 6803 (hereafter PCC 6803) . Ph. Ph41]. The ΔZ and cysteine mutant cell lines showed altered pigment abundance and slower growth rates compared to the controls. Therefore, we reasoned that they could exhibit altered light harvesting or energy transfer phenotypes. We conducted 77 K fluorescence spectroscopy with excitation at either 440 nm to preferentially excite chlorophyll or 580 nm to preferentially excite phycocyanin. Fluorescence emission spectra (600–800 nm) containing peaks originating from terminal emitters of phycocyanin (PC), photosystem II (PSII), and photosystem I (PSI) were then collected . ChloropBulk fluorescence and growth experiments revealed altered pigment profiles and reduced growth capacity of ∆Z compared to the WT. The growth and fluorescence properties of ∆Z and a WT control strain expressing GFP (WT-GFP) were also compared at single(sub)-cellular resolution using long-term time-lapse fluorescence microscopy  on a cusE. coli, Rnr, Rnb, and polynucleotide phosphorylase (PNPase) work cooperatively and redundantly to provide the cell with 3′-5′ exonuclease activity. Deletion of any one of those proteins produces only mild phenotypes. However, deletion of PNPase in concert with either Rnr or Rnb is lethal . T,4. Tzam This work provides new insight into the RNase II/R family of cyanobacteria. We classify Zam proteins as non-functional nucleases and show that they have a role in controlling diverse phenotypes including growth rate, pigment biosynthesis, and the PSI:PSII ratio. The ability of Zam to function in vivo is dependent upon redox active cysteines located in CSD1, and this highly conserved region is almost certainly responsible for controlling the function of Zam. While further experimentation is needed, a model where the redox state of Zam modulates both the interaction with and subsequent activation of RNase E seems likely based on our work and the literature.


Independent Component Analysis (ICA) allows the dissection of omic datasets into modules that help to interpret global molecular signatures. The inherent randomness of this algorithm can be overcome by clustering many iterations of ICA together to obtain robust components. Existing algorithms for robust ICA are dependent on the choice of clustering method and on computing a potentially biased and large Pearson distance matrix.robustica, a Python-based package to compute robust independent components with a fully customizable clustering algorithm and distance metric. Here, we exploited its customizability to revisit and optimize robust ICA systematically. Of the 6 popular clustering algorithms considered, DBSCAN performed the best at clustering independent components across ICA iterations. To enable using Euclidean distances, we created a subroutine that infers and corrects the components’ signs across ICA iterations. Our subroutine increased the resolution, robustness, and computational efficiency of the algorithm. Finally, we show the applicability of robustica by dissecting over 500 tumor samples from low-grade glioma (LGG) patients, where we define two new gene expression modules with key modulators of tumor progression upon IDH1 and TP53 mutagenesis.We present robustica brings precise, efficient, and customizable robust ICA into the Python toolbox. Through its customizability, we explored how different clustering algorithms and distance metrics can further optimize robust ICA. Then, we showcased how robustica can be used to discover gene modules associated with combinations of features of biological interest. Taken together, given the broad applicability of ICA for omic data analysis, we envision robustica will facilitate the seamless computation and integration of robust independent components in large pipelines.The online version contains supplementary material available at 10.1186/s12859-022-05043-9. Independent Component Analysis (ICA) is a matrix factorization method that dissects a mixture of signals into a predefined number of additive independent sources or components. ICA finds sets of statistically independent components by minimizing their mutual information . In biolFastICA [Icasso to address the inherent randomness of FastICA, by running FastICA multiple times and clustering the components of source matrices across all runs  and found two sets of genes related to the mechanisms by which mutations in IDH1 and TP53 lead to tumor progression.Here, we developed it-learn . By leverobustica is written in Python under the open-source 3-Clause BSD license. The source code and documentation are freely available at https://github.com/CRG-CNAG/robustica. Additionally, all scripts to reproduce the work presented are available at https://github.com/MiqG/publication_robustica.RobustICA. After instantiation, one can use the fit method on a data matrix to run the FastICA algorithm multiple times and cluster the resulting independent components with the desired number of components and clustering distance metric and algorithm. Subsequently, one can recover the computed source and mixing matrices through the transform method.The algorithm to carry out robust ICA is controlled by the main class FastICA class already available in scikit-learn. For this reason, we only required 6 more arguments labeled using the “robust_” prefix. These arguments determine: the number of times to run FastICA before clustering (robust_runs), whether to use our subroutine to infer and correct the signs of the components across FastICA runs (robust_infer_signs), the custom clustering algorithm class (robust_method), the keywords to pass to the clustering algorithm class (robust_kws), whether to speed up the clustering step using PCA to reduce the dimensions of the independent components across all runs (robust_dimreduce) and, when the distance needs to be precomputed, the function to use to compute pairwise distances in the clustering step (robust_precompdist_func).To facilitate the customizability and seamless integration of the algorithm, the user can specify all the parameters related to every step of the robust ICA algorithm as similarly as possible to the core robustica, one can fully customize the clustering method to use as long as they follow scikit-learn conventions [AffinityPropagation as it did not converge for any of the datasets using its default parameters.With ventions . We purpk (k-based) or a free (density-based) number of clusters. Overall, k-based clustering algorithms -AgglomerativeClustering, KMedoids- were fast and memory-efficient but resulted in lower silhouette scores than density-based algorithms -CommonNNClustering, DBSCAN, OPTICS- . As expected, the number of initial samples used to run ICA multiple times is not associated with any performance metric, and memory usage and silhouette scores increased as we dissected datasets with more features  . In addition, we compressed the feature space of all ICA runs through PCA to reduce the overall run time and memory usage while maintaining the same performance  using different clustering metrics. Our sign inference-and-correction subroutine combined with Euclidean distances produced a total of 94 robust independent components, 31 more than Pearson distances with a similar overall quality and computational efficiency  and tumor protein P53 (TP53) are highly common among the 530 LGG tumor samples analyzed , identified either through both clustering metrics or only through Euclidean distances, respectively. From the properties considered, weights in component 72 were highly associated with IDH1 mutation status, expression-based indices of cell proliferation, and patient overall survival probability  and was enriched with 98 targets of E2Fs  and 159 (module 12) genes using the extreme weights of the corresponding component (Additional file MKI67 . Interesrobustica and our revisited Icasso algorithm to identify gene sets whose unique transcriptional signature in LGG is associated with genotypes and phenotypes of interest, demonstrating the biological applicability of this approach (Fig. With this, we demonstrate the utility of ach Fig. D.robustica, a new Python package built on top of scikit-learn that enables performing precise, efficient, and customizable robust ICA seamlessly. Using different clustering algorithms and distance metrics, we tested whether robust ICA could be further optimized. Our sign correction subroutine improved the resolution, robustness, and computational efficiency of the clustering step. As an example, we explored how the gene modules generated with robustica from transcriptomic profiles of LGG patients are associated with multiple markers of the disease’s progression simultaneously. Overall, robustica makes high-performance robust ICA more accessible to potentially analyze any omic data type and facilitates its incorporation in larger computational pipelines.We created Additional file 1. Document containing Methods, Supplementary Figures, and Supplementary Tables.