Drosophila melanogaster using isogenic lines with heritable variation in both male and female traits known to influence competitive fertilization. We confirmed, and expanded on, previously reported genotypic interactions within and between the sexes, and showed that several reproductive events, including sperm transfer, female sperm ejection, and sperm storage, were explained by two‐ and three‐way interactions among sex‐specific phenotypes. We also documented complex interactions between the lengths of competing males’ sperm and the female seminal receptacle, which are known to have experienced rapid female‐male co‐diversification. Our results highlight the nonindependence of sperm competition and cryptic female choice and demonstrate that complex interactions between the sexes do not limit the ability of multivariate systems to respond to directional sexual selection.How males and females contribute to joint reproductive success has been a long‐standing question in sexual selection. Under postcopulatory sexual selection, paternity success is predicted to derive from complex interactions among females engaging in cryptic female choice and males engaging in sperm competition. Such interactions have been identified as potential sources of genetic variation in sexually selected traits but are also expected to inhibit trait diversification. To date, studies of interactions between females and competing males have focused almost exclusively on genotypes and not phenotypic variation in sexually selected traits. Here, we characterize within‐ and between‐sex interactions in  For species with internal fertilization and female promiscuity, postcopulatory sexual selection (PSS) is believed to depend, in part, on complex interactions between rival males and between the sexes. Although little investigated, clarifying such interactions is critical as they may limit the efficacy of PSS in the diversification of reproductive traits . Here, we resolve how sex‐specific traits and their interactions contribute to key reproductive events and outcomes related to competitive fertilization success, including traits known to have experienced rapid diversification. Our results provide novel insights into the operation and complexity of PSS and demonstrate that the processes of sperm competition and cryptic female choice are not independent selective forces. Simultaneously, the complex interactions between the sexes do not necessarily limit rapid trait diversification in multivariate systems.Because females of most species mate with multiple males within reproductive cycles  and of discriminating between competing sperm  by using artificial insemination , or else have examined only a single pair of interacting, sex‐specific traits, such as sperm length and female seminal receptacle (SR) length (Miller and Pitnick D. melanogaster with sperm that express either green (GFP) or red (RFP) fluorescent protein in their sperm heads  identifying many of the putative sex‐specific targets of PSS , (2) quantifying their genetic variation, and (3) determining their contribution to variation in competitive fertilization success within a multivariate framework. To accomplish these goals, we embarked on a three‐stage research program using isogenic populations of  Civetta , 2013, fm populations of D. melanogaster that express a protamine labeled with either GFP or RFP in sperm heads  in D. melanogaster (including in these isogenic lines) is directly proportional to the respective numbers of sperm from two competing males remaining in storage (S2), particularly within the SR, after females have ejected any excess second‐male and displaced first‐male sperm, thus following a fair raffle among stored sperm . Females not remating within 4 hours were given additional 4‐hour remating opportunities on days 3–5 after the first mating. Immediately after the end of the second mating, we removed the male from the mating vial, isolated the female in a glass three‐well spot plate beneath a glass coverslip, and checked for sperm ejection every 10 min for up to 5 hours using a stereomicroscope. We recorded the time to sperm ejection, immediately removed the female from the well and froze it for later quantification of stored sperm, and transferred the ejected mass to phosphate‐buffered saline (PBS) on a microscope slide and sealed the coverslip with rubber cement.Within each of eight replicates, examined in four temporal blocks of two full replicate sets (staggered by 2 days), we mated virgin females each to an RFP male and, 2 days later, to a GFP male in all 108 possible combinations between genotypes  across all storage organs and within the SR, respectively. Combining these counts with those of the ejected masses further permitted calculating the number of first‐male resident sperm at the time of remating, the number of second‐male sperm transferred, and both the absolute and relative number of each male's sperm stored and ejected, respectively.For all dissected females, we counted the sperm of both competitors across the different organs of the FRT  and determined the total number of sperm for each male in all female sperm‐storage organs combined, and the proportion of total sperm derived from the second male , rinsed it with PBS, and mounted it under a coverslip in glycerol and PBS (80:20 [v/v]). We measured the length of five sperm per male using the segmented line tool in ImageJ version 1.47 at 200× magnification under the dark‐field optics of an Olympus BX‐60 microscope. For each of the six female genotypes, we measured the thorax length of each of eight females, and dissected their reproductive tract into PBS on a microscope slide and covered it with a glass coverslip with clay at the corners, allowing the SR to be flattened to two dimensions without stretching. We then measured SR length using ImageJ at 200× magnification under an Olympus BX‐60 microscope with Nomarski DIC optics. Both sperm and SR length are significantly heritable  with a binomial error distribution, a logit link, and an additional observation‐level random effect to account for overdispersion. Each model included the female and first‐male and second‐male genotypes with all two‐ and three‐way interactions as fixed effects, except where only females and first males and their interaction were predicted a priori and so any second‐male contributions were excluded. We used conditional F tests (LMM) or Wald χ2 tests (GLMM) to test for significant main and interactive genotypic effects.We performed all analyses using the statistical software package R version 3.4.3  of any model with a lower AICc value , we examined the interrelationships between the male and female reproductive traits themselves. Due to their complexity and a lack of specific information on how precisely traits should interact to explain focal traits, these analyses were necessarily somewhat exploratory. Thus, instead of null‐hypothesis significance testing based on a priori predictions, we used an information‐theoretic approach to account for model uncertainty and identify the most plausible model(s)  averages and 95% confidence intervals of their coefficients as well as their relative variable importance across the confidence model set (Burnham and Anderson piecewiseSEM (Lefcheck 2 in SR) as a proxy of fitness outcomes , and particularly any two‐ or three‐way interactions between them, contributed to variation in reproductive parameters , using (G)LMMs with the four temporally separated blocks as a random factor. We omitted in all analyses females that did not complete the experiment (death or loss), had no recorded remating by day 5, or post hoc showed no evidence of successful sperm transfer during the first mating . These exclusions reduced our sample size from 864 to 744 successful mating trios, but additional missing data  resulted in varying sample sizes between analyses.2  and to the fertilization set   and a weak trend for a three‐way interaction . The fertilization set was influenced by a three‐way interaction , a male × male interaction , and a first‐male main effect . Rather, the female remating interval, ranging between 2 and 5 days, was explained solely by the female genotype . Further, controlling for the remating interval and omitting the second‐male genotypes due to no a priori expectation of their contribution, the number of progeny produced between the first and second mating was influenced primarily by the female genotype , with a weak contribution of the first‐male genotype . Similarly, accounting for the number of progeny produced as a proxy of sperm use, the number of first‐male sperm still residing in the FRT at remating was also explained by both the female  and first‐male main effects .Next, in LMMs, we focused on two‐ and three‐way interactions between members of the mating trio on specific reproductive traits predicted to contribute to SF5,630.50 = 55.05, P < 0.0001), but also by a female × first‐male interaction . The number of sperm transferred during these copulations was explained by a trend for a three‐way interaction between all individuals of a mating trio  in addition to a strong second‐male main effect . Finally, the time to female sperm ejection after remating was determined by the female  and second‐male genotypes  and their interaction .All later reproductive stages exhibited at least some interactive effects, albeit no statistically significant three‐way interaction. For example, the duration of the second copulation was explained primarily by the second‐male's genotype , represented by strong positive effects of copulation duration  and the number of resident sperm (β = 0.48 [0.32–0.63]), and a weak trend for an interaction between them . These results suggest that males transfer more sperm when there are numerous resident sperm in the FRT, by prolonging copulation. Next, we tested the prediction that the time to female sperm ejection should be influenced by the joint effects of SR length and the differences  in sperm length and number between males (N = 529). Here, only the difference in sperm length had an effect . Further examination using only the absolute sperm lengths of both males rather than the difference between them revealed that this effect was driven primarily by the second male's sperm length . This result suggests that longer second‐male sperm might prolong the time to female sperm ejection and thus the sperm displacement phase, which was previously shown to increasingly bias sperm storage toward the second male  should be explained by the relative sperm lengths and the numbers of first‐male resident and second‐male transferred sperm at the end of copulation  including all interactions between ejection time and the between‐male differences in sperm length and numbers, respectively, and with a binomial error distribution and logit link as well as an observation‐level random effect to account for overdispersion, total S2 increased with both ejection time (β = 0.43 [0.27–0.60]) and the difference in sperm numbers (β = 0.69 [0.51–0.86]), and was further influenced by an interaction between these two predictors . Although the full model set contained 1294 different models, this was reduced to only 22 models (Table S12) by removing models that were simply more complex versions of any model with a lower AICc value , SR length (β = –0.56 [–0.79 to –0.32]), the time to sperm ejection (β = 0.87 [0.63–1.11]), and difference in the number of sperm between males (β = 0.76 [0.51–1.00]) being the most important predictors. The difference in sperm length appeared unimportant as a main effect but contributed to all three interactions whose 95% confidence interval excluded zero after model averaging .Finally, we used a piecewise structural equation modeling approach to better visualize both direct and indirect effects of these numerous male and female traits on one another, and particularly on the fertilization set. This analysis revealed a complex network of interrelationships Fig. , 2013. I1 genotypes for all traits examined, and we had only few genotypes, we expect the “traits analyses” to be more sensitive to detecting interactions than the genotypic analyses.There is a growing paradigm in sexual selection theory that emphasizes interactions between the sexes  among genotypes, we found that a three‐way interaction between the different genotypes of a mating trio significantly contributed to variation in the fertilization set, which is a consequence of all other traits examined  and arguably the strongest correlate of variation in male and female fitness  and cryptic female choice . Moreover, sperm length, which is recognized as a PSS ornament that interacts functionally and evolutionarily with SR length .All data are deposited in the Dryad Repository .Supplementary Table S2: Results of a generalized linear mixed model with binomial error distribution representing the genotypic effects on S2 within the seminal receptacle  after female sperm ejection .Supplementary Table S3: Results of a linear mixed‐effects model  analyzing the genotypic effects and interactions on the female remating interval.Supplementary Table S4: Results of a linear mixed‐effects model  analyzing the genotypic effects and interactions on the number of offspring produced between copulations, controlling for the female remating interval.Supplementary Table S5: Results of a linear mixed‐effects model  analyzing the genotypic effects and interactions on the number of 1st‐male sperm residing in the female reproductive tract at remating, controlling for the number of progeny produced prior to remating.Supplementary Table S6: Results of a linear mixed‐effects model  analyzing the genotypic effects and interactions on the duration of the second copulation.Supplementary Table S7: Results of a linear mixed‐effects model  analyzing the genotypic effects and interactions on the number of sperm transferred by the second male.Supplementary Table S8: Results of a linear mixed‐effects model  analyzing the genotypic effects and interactions on the time to female sperm ejection.Supplementary Table S9: Results of the information‐theoretic analyses examining the effects of copulation duration (C), the number of 1st‐male sperm residing in the FRT at remating (R) and female thorax length (T) on the number of sperm transferred by the second male (N = 557).Supplementary Table S10: Results of the information‐theoretic analyses examining the effects of the difference in sperm length (L), the difference in sperm number (N) and seminal receptacle length (S) on the time to female sperm ejection (N = 527).Supplementary Table S11: Results of the information‐theoretic analyses examining the effects of the difference in sperm length (L), the difference in sperm number (N) and the time to female sperm ejection (E) on the relative numbers of sperm stored between males .Supplementary Table S12: Results of the information‐theoretic analyses examining the effects of the difference in sperm length (L), the difference in sperm number (N), the time to female sperm ejection (E), female SR length (S) and female thorax length (T) on S2 within the SR (N = 508). Each model was a GLMM with the temporal block as a four‐level random factor. An observation‐level random effect was included to account for overdispersion.Click here for additional data file.


Lactate measurements have proven utility as a triage tool, therapeutic guide, and prognostic indicator, with broad use in Acute Care and transplantation. Its value in guiding therapy and predicting outcomes following liver resection is less well‐defined. This systematic review in accordance with Preferred Reporting Items for Systematic Reviews and Meta‐Analyses guidelines assessed the relationship between peri‐operative lactate levels and morbidity and mortality after liver resection. Seven relevant studies comprising 2573 patients in total were identified. Six studies assessed intra‐operative or early postoperative lactate levels, one publication examined pre‐operative levels. All studies demonstrated a significant association between peri‐operative lactate levels and adverse outcomes. The influence of pre‐operative diabetes and cirrhosis on postoperative lactate levels was shown in one study each, no study assessed the association of lactate with post‐hepatectomy liver failure according to defined criteria. The heterogeneity of study measurements and end‐points precluded a meta‐analysis from being performed. Early postoperative lactate >3‐3.7 mmol/L is associated with mortality but validation of clear cut‐off levels for outcome prediction is pending. Literature suggests lactate is a useful predictive marker for outcomes post liver surgery, especially when measured in the early postoperative phase. Further research is required to standardize the use of lactate measurements in a meaningful therapeutic manner. HighlightIn this systematic review of seven studies analyzing pre‐, intra‐ or postoperative lactate levels in 2573 patients, Connolly et al demonstrated that lactate levels at all time‐points were associated with post‐hepatectomy outcomes, and early postoperative lactate predicted mortality. Possible clinical applications include adaption of intraoperative strategies and intensification of postoperative management. Some examples include the American College of Surgeons National Surgical Quality Improvement Program (ACS‐NSQIP) and the Physiological and Operative Severity Score in the enUmeration of Mortality and Morbidity (POSSUM).Lactate measurements have demonstrated a strong correlation to clinical outcomes in critically ill patients, and are now a keystone criterion in the diagnosis of shock.The aim of this systematic review was to evaluate whether peri‐operative lactate measurement provides a simple yet accurate prediction of post‐hepatectomy outcomes.22.1A systematic literature search was conducted to investigate the relationship between peri‐operative lactate levels and postoperative complications in patients undergoing liver surgery. The search was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines, using PubMed, EMBASE, Medline, and Cochrane databases.2.2The search results were pooled into a citation manager, and duplicates were removed. Abstracts of the remaining studies were screened by two investigators independently to extract relevant studies. The full texts of the relevant studies were then examined, and selections were refined accordingly. The reference lists of the selected studies were also examined to identify additional publications fulfilling the inclusion criteria. Any discrepancies were resolved through discussion and subsequent consensus. The data extracted from the studies included: study design; number of patients and patient characteristics; time‐point and method of lactate measurement; operative details; and outcomes measured. All studies were graded for methodological quality using the Newcastle‐Ottawa quality assessment scale.33.1The initial search strategy yielded a total of 495 records. Following application of the inclusion criteria and removal of duplicates Figure  seven stSix studies evaluated postoperative mortality and five studies assessed overall postoperative morbidity.3.2Methodological assessment details according to the Newcastle‐Ottawa quality assessment scale are summarized in Table 3.3The median age of the cohorts varied from 59 to 68 years. Four of the studies documented information regarding the pre‐operative status of the patients, for example in the form of their American Society of Anesthesiologists (ASA) grade or Charlson Comorbidity Index (CCI) score.3.4The indications for surgery across all studies included primary and secondary liver malignancies, benign tumors, trauma, and parasitosis. One study did not specify operative pathology; however, the remaining six studies all reported liver metastases as the most prevalent diagnosis.3.5Whilst all patients underwent liver resections, one study excluded laparoscopic procedures and a further study included extended hepatectomies only.3.6Table P < .001).The retrospective study published by Watanabe et al in 2007 (n = 151) demonstrated a significant difference in the mean initial postoperative lactate level between survivors and non‐survivors (4.1 mmol/L vs 10.1 mmol/L).Wiggans et alIn the study by Meguro et al,The study by Pagano et alRiediger et alIn response to a systematic review by Lim et al which evidenced the poor accuracy of risk prediction models for liver resections, Vibert et al sought to build and subsequently validate a new prognostic model.Finally, in the study by Lemke et al,4All studies in this systematic review independently demonstrated the usefulness of peri‐operative lactate measurements for predicting outcomes following liver surgery.One study in this review examined the prognostic value of pre‐operative lactate levels on postoperative outcomes, whereas the remaining six studies measured either intra‐operative or early postoperative levels.In the large prospective observational study by Vibert et al,Lactate has been established as useful marker in Emergency and Intensive Care settings, both as a diagnostic tool for septic shock, and as a therapeutic end‐point to guide fluid resuscitation.In addition to the uncertainty over what constitutes an appropriate lactate cut‐off level post‐hepatectomy, there is also the question of how clinicians respond to the perceived elevated levels. Hyperlactatemia can be contributed to via several mechanisms including tissue hypoxia, anaerobic metabolism, microcirculatory dysfunction, and reduced clearance ability.Effective lactate clearance has been associated with better patient outcomes across a number of studies on Emergency and Intensive Care patient cohorts.A key limitation to this systematic review was that most studies were found to be of moderate methodological quality. In particular, many studies were inherently biased as they included only those patients who had lactate tested peri‐operatively.In summary, being aware of the aforementioned limitations, from a clinical perspective the results of this systematic review might be translated into daily surgical care in several different scenarios. First of all, markedly increased pre‐operative lactate levels  may preclude patients from undergoing elective liver surgery, especially in case of concomitant presence of high serum bilirubin, since their expected risk for postoperative mortality exceeds 23%.Secondly, early raised postoperative lactate values are clearly associated with increased mortality and morbidity, and they might therefore serve as a stratification tool for intensified postoperative observation on intensive care units or prophylactic measures against infections, liver failure or bleeding .Thirdly, intra‐operative measurements of lactate dynamics could theoretically facilitate adaption of surgical strategies in real‐time. For example, in complex cases requiring extensive resections plus a reconstructive phase (particularly hilar cholangiocarcinoma), an excessive increase in intra‐operative lactate values might even justify discontinuation of the procedure after parenchymal resection, temporal extracorporeal drainage of bile fluids through percutaneously placed surgical drains and final two‐stage completion with a biliary‐enteric reconstruction within 24–48 hours. Ideally, future studies designed to answer some of these questions should be performed prospectively and with international validation, include all patients undergoing minor and major liver resection, evaluate specific complications such as PHLF and collect data on various time‐points of lactate measurements to allow for analysis of lactate dynamics and determination of fixed and well‐defined time‐points for standardized use.To conclude, while the heterogeneity of the studies meant that a meta‐analysis was not feasible, it is promising that all studies reached a positive conclusion on the usefulness of lactate for predicting outcomes post liver surgery. Further research is required to understand the strength of this correlation, the underpinning pathophysiological mechanisms, and to provide evidence‐based guidance for clinical decision‐making. There is also a need for future studies to address a particular gap in the literature pertaining to lactate and the prediction of PHLF as well as potential therapeutic intervention.All authors declare no conflict of interest for this article.All authors have contributed to design of the study, analysis of the data and drafting/revision of the manuscript, have approved the final article and are accountable in all aspects of the work.


A novel suspended, modular, and 3D‐printed total sacral implant had been used for reconstruction. This series included two female and three male patients, with a mean age of 42.2 years . Surgical time, blood loss, complications, preoperative and postoperative neurological function, instrumentation failure, and local control were presented and analyzed.Five patients who had undergone total piecemeal resection of SGCT involving upper sacral segments  and the mean operative blood loss 4400 mL . The mean follow‐up was 15 months. After the operation, pain was significantly relieved, and the patients resumed walking as early as 2 weeks later. The patients showed no neurogenic bladder dysfunction and no fecal incontinence or gait disturbance. Wound healing was poor in one patient. Patients recovered well without evidence of local recurrence. No implant failures or related clinical symptoms were detected during follow up. Satisfactory bone ingrowth and osseointegration at the bone‐implant junctions was found in follow‐up CT.All patients underwent the operation without death or serious complications. The implant was installed on the defect, connecting the ilium and lumbar vertebrae, and fixed with a screw–rod system up to the level of L1–3 nerve roots in patients with SGCT. We believe that this implant can be applied to sacral reconstruction in a wide variety of diseases.Although technically challenging, it is feasible and safe to use a suspended, modular, and 3D‐printed implant for reconstruction after total piecemeal resection with the preservation of bilateral S Giant cell tumor of the bone is a locally aggressive benign bone tumor that rarely metastasizes to the lungs, with malignant transformation occurring in fewer than 1% of all giant cell tumors of the bone (GCTB)1 vertebra often requires spinopelvic reconstruction. When S1 remains incomplete after resection, weight‐bearing capacity is considered insufficient and reconstruction needs to be performed. Spinopelvic reconstruction is challenging in terms of anatomical complexity, excessive load, and extensive defects, and there is a high risk of complications. The pedicle iliac screw rods system with human bone or titanium mesh is typically used to rebuild biomechanical stability of the spinopelvic complex. The previous spinopelvic reconstruction method is often unable to reconstruct defects satisfactorily. Mechanical failure, such as rod fatigue fracture and loosening, is sometimes encounteredSurgery for giant cell tumors involving the S1‐3 nerves via a posterior‐only approach. To our knowledge, there are no previous case series reports of successful spinopelvic reconstruction using a 3D‐printed total sacral implant in patients with SGCT after tumor resection.The technological advances in 3D‐printing may help us overcome these difficulties. 3D‐printed technology has been successfully used in bone tumor surgery during preoperative planning, resection, and reconstruction1–3 nerves via a posterior‐only approach between September 2017 to July 2018 were retrospectively reviewed. Suspended, modular, and 3D‐printed total sacral implants were used for reconstruction. There were three men and two women in this case series, with a mean age at the time of diagnosis and admission of 42.2 years . Informed consent was obtained from all individual participants included in the study. The protocol for the research project was approved by the Medical Ethics Committee of Qilu Hospital of Shandong University and it conforms to the provisions of the Declaration of Helsinki (as revised in Brazil in 2013). Patient characteristics and outcomes are provided in Table Five patients who had undergone total piecemeal resection of SGCT involving upper sacral segments and the midline with the preservation of bilateral S1 vertebrae and below. S1–4 were involved in two patients; S1–3 were involved in three patients; MRI revealed presacral soft tissue masses in four patients.All patients underwent plain radiography, CT, and MRI to identify the form, location, and size of the tumor and its relationship with surrounding structures. All tumors were located in the SA preoperative biopsy was planned to obtain tissue for a pathological diagnosis after imaging studies. Percutaneous CT‐guided needle biopsy is the preferred diagnostic technique in these cases, not only yielding adequate tissue for diagnosis but also minimizing the possibility of tumor cell contamination. All patients underwent CT‐guided needle biopsy and histologic examination showed giant cell tumors of the bone.Denosumab was administered subcutaneously at a dose of 120 mg twice a month before surgery under oral supplementation of calcium and vitamin D. Subjective symptoms were relieved after treatment of neoadjuvant denosumab in all five patients. The tumors were reduced in size and clear in the boundary.5 inferior endplate and a right wing‐part designed to connect the right ilium osteotomy plane and the right iliac crest. The second module  and the mean operative blood loss was 4400 mL .All five patients underwent total piecemeal resection of SGCT with preservation of the bilateral SThe excised specimens were sent to the pathology department for histologic analysis. The pathologic diagnoses were GCTB.There were no intraoperative complications. Poor wound healing occurred in one patient, who recovered completely after 2 weeks’ management with debridement and dressing change. The average duration of follow‐ up was 17 months, with a range of 13–21 months. No local recurrence or instrumentation failure was detected during follow up. After the operation, the pain was significantly relieved, and the patients resumed walking as early as 2 weeks later. The patients showed no neurogenic bladder dysfunction, fecal incontinence, or gait disturbance. Patients recovered well during the follow‐up period. Satisfactory bone fusion was found in CT  complained of low back pain for 1 month. Anteroposterior radiographs of the sacrococcygeal area showed a large area of osteolytic destruction at the S3 nerve and above is necessary to maintain good mental health, physical health, bowel function, and sexual functionet al. treated SGCT with intralesional curettage or partial excision and the 5‐year local recurrence rate was 20.4%3 nerve roots and above were preserved and no patients had experienced local recurrence during the follow up. The neurologic outcome of the present study is in accordance with the results reported in the literatureSurgical treatment remains the mainstay of treatment for SGCT, and recurrence is a major concern in treatment, likely because of the complex location and the large size before diagnosis1 and S2) and the midline often results in a disconnection between the pelvis and the lumbar spine that is similar to total sacrectomy. Total sacrectomy without reconstruction is associated with some problems in patients, such as being bedridden long term, nerve root distraction from the submergence of lumbar vertebrae, and walking disability. Therefore, spinopelvic reconstruction after total sacrectomy is recommended by several authorset al. reported that postoperative fixation mechanical failure occurred in 25% of patients (16/63) who underwent total sacrectomyThe bone defect caused by the total piecemeal resection of SGCT involving upper sacral segments (SThis novel suspended, modular, and 3D‐printed total sacral implant can overcome the limitations. It integrates the three categories mentioned above simultaneously, which shortens the operation time and simplifies the procedure. The structure can suit the complex and high‐strength mechanical environment of the lumbosacral region. The modular design prevents nerve root damage in implant placement, avoiding excessive traction of the nerve roots. It also allows fine‐tuning when the implant does not match the actual bone defect precisely. The suspended design of the implant and screw direction in accordance with the direction of mechanical transmission enables the best mechanical distribution. The combination of a cancellous bone screw and a locking screw ensures the tight fit of the bone–implant junctions, ensuring the short‐term stability. By reducing the gap between the implant and the bone and increasing the contact area, tightly bonded implants can better maintain stability and reduce postoperative pain. The design of foramen around the implant is beneficial to the suture and fixation of soft tissue. The bone–implant junctions of the 3D‐printed implant are porous structures, which are beneficial to bone ingrowth and osseointegration, ensuring long‐term stability. The follow‐up results showed that excellent bony union can be achieved on both the densely structured strut surface and the loosely structured porous mesh.Denosumab, a fully human monoclonal antibody that inhibits RANKL, was approved by the Food and Drug Administration in 2013 and represents a new treatment option for GCT. The effect of preoperative denosumab therapy was confirmed for patients with unresectable GCTB and those with GCTB who needed surgical downstaging because surgery would be associated with severe morbidityThe present study has some limitations. First, this was a retrospective study, although it is, to our knowledge, the largest series to data on 3D‐printed total sacral implants for reconstruction after resection of SGCT. Second, the total number of subjects is small because of the relative rarity of SGCT, and this limits the power of statistical analysis and tests. Third, our follow up is too short to provide data concerning long‐term tumor control. In future studies, these findings may need to be confirmed with larger populations from multiple centers.1–3 nerve roots. Considering the complexity of the surgery, we recommend that this surgery should be conducted by an experienced team with multidisciplinary cooperation and careful preoperative planning, which are key to success. We believe that this implant can be an effective means of managing sacral reconstructions in a wide variety of diseases.The suspended, modular, and 3D‐printed total sacral implant is a reasonable choice for reconstruction after total piecemeal resection of SGCT with the preservation of bilateral S


Code and data for reproducibility, as well as an interactive environment to explore the study data, can be accessed at https://github.com/jsheunis/quality‐and‐denoising‐in‐rtfmri‐nf.Neurofeedback training using real‐time functional magnetic resonance imaging (rtfMRI‐NF) allows subjects voluntary control of localised and distributed brain activity. It has sparked increased interest as a promising non‐invasive treatment option in neuropsychiatric and neurocognitive disorders, although its efficacy and clinical significance are yet to be determined. In this work, we present the first extensive review of acquisition, processing and quality control methods available to improve the quality of the neurofeedback signal. Furthermore, we investigate the state of denoising and quality control practices in 128 recently published rtfMRI‐NF studies. We found: (a) that less than a third of the studies reported implementing standard real‐time fMRI denoising steps, (b) significant room for improvement with regards to methods reporting and (c) the need for methodological studies quantifying and comparing the contribution of denoising steps to the neurofeedback signal quality. Advances in rtfMRI‐NF research depend on reproducibility of methods and results. Notably, a systematic effort is needed to build up evidence that disentangles the various mechanisms influencing neurofeedback effects. To this end, we recommend that future rtfMRI‐NF studies: (a) report implementation of a set of standard real‐time fMRI denoising steps according to a proposed COBIDAS‐style checklist ( It was initially proposed and developed by Cox, Jesmanowicz, and Hyde  has gained much interest in the past decade due to its ability to help subjects achieve learned regulation of regional brain activation, as was initially demonstrated by Yoo and Jolesz  could learn to regulate the BOLD response in the rostral anterior cingulate cortex, leading to significant changes in pain perception in this group.N = 114 patients). This apparent inability to replicate pilot findings in a larger sample size, which featured as a prominent discussion at the first Swiss rtfMRI Neurofeedback Conference  of the rtfMRI‐NF signal and of real‐time fMRI data and studies in general. Specifically, we investigate three research questions:To preface addressing these questions, a background on the BOLD signal and its confounds and on the details of the calculated neurofeedback signal is provided. Although both acquisition and processing methods are covered in this work, focus is given to the latter. We conclude with a general discussion and future recommendations based on the reviewed literature.22.1The noisy BOLD signal: The T2*‐weighted BOLD signal typically acquired using standard gradient‐echo echo‐planar imaging (EPI) in fMRI represents hemodynamic and metabolic responses, through a neurovascular coupling, to alterations in neuronal activity , it should be no surprise that the BOLD signal has several confounds and remains difficult to isolate as a proxy for true neuronal activity  fMRI noise, confounds and artefacts. Head motion, for example, has been shown to result in false activity patterns when coupled to the timing of the task paradigm  as the preferred acquisition method, likely due to its prevalence in conventional functional imaging. Acquired slices are reconstructed on the MRI scanner hardware, and upon completion, each functional image volume is typically exported and shared on a local network from where it is accessible by the real‐time fMRI application software. Figure Time frame definitions: The ‘real‐time’ time frame is loosely defined to be the interval between two successive functional image scans, that is, the repetition time (TR), indicating that the concept ‘real‐time’ varies according to the application. Ideally, all required reconstruction, export and processing steps for each functional image should be completed sufficiently prior to or by the time the next image in the session is acquired, thus allowing the presentation of up‐to‐date image information to the researcher and/or subject. Given the nature of the HRF, real‐time fMRI not only includes a delay of one TR typically used for data processing (reported to be ~1–3 s in standard rtfMRI‐NF applications), but also a substantial delay due to the indirect measurement of neuronal activity (~4–6 s). As such, typical implementations of real‐time fMRI often only allow a representation of brain activity about 5 s or more after such changes occurred on a neuronal level, leading to the term ‘near‐real‐time’. This definition is distinct from the same term used by some studies to refer to a real‐time fMRI processing stream that delivers brain activity and other information within minutes after completing the functional scan session  and to the inclusion of the minimum sufficient steps to achieve an acceptable level of data quality, so as to decrease per‐volume processing time. This trade‐off between maintaining a high level of method accuracy and minimising the required per‐volume processing time has initially been a large constraint to expanding the complexity of real‐time fMRI processing steps, but has become increasingly easier to manage with advances in modern computing technology and algorithm development. The use of parallel computing using clusters  BOLD activity percentage signal change typically in a single or differential region of interest (PSCNef), (b) functional connectivity between BOLD activity in multiple ROIs (FCNef) and (c) multivariate (or multivoxel) pattern analysis (MVPA), typically within a single ROI (DecNef).Percentage signal change neurofeedback: The majority of volunteer and patient rtfMRI‐NF studies have used a single or multiple ROI approach to calculate the feedback signal, specifically using the percentage signal change (PSCNef) of the spatially averaged signal obtained from all voxels within the defined ROI(s), as illustrated in Figure Functional connectivity neurofeedback: FCNef  are either absent from or not biasing our interpretation of the processed data, and there is lower possibility of making false inferences. This improves results and inferential reproducibility, and thus scientific progress. As an extension of fMRI data, high quality of the real‐time fMRI neurofeedback signal implies that a signal closely reflective of brain activity (and not of noise or artefacts) is fed back to the subject in real‐time. Quality in methods reporting implies that a published study contains enough information about the applicable experimental‐, acquisition‐ and data processing steps that would allow different researchers to reproduce the methods. Here, high quality has a direct and beneficial influence on methods reproducibility.Quality is an umbrella term that is applicable to real‐time fMRI data, to the neurofeedback signal and to the methods reporting process. Generally, fMRI data quality is a measure of how well the acquired BOLD data reflects the signal of interest, that is, neural activity, and it is influenced by variability in multiple factors including the subject, the experimental design and acquisition . If fMRI data quality is high, the implication is that signals that are When aiming to improve quality in real‐time fMRI neurofeedback it is therefore advised to (a) separate the effects of noise  from true BOLD fluctuations, (b) quantify and report the quality of real‐time fMRI data and the calculated neurofeedback signal and (c) accurately and sufficiently report the use of applicable real‐time fMRI denoising methods.3.1Traditionally, apart from expert visual inspection of fMRI datasets to identify low quality volumes/sessions/subjects/sites , the temporal signal‐to‐noise ratio (tSNR) has been an important quantitative measure of fMRI data quality and the ability of an experiment to find effects of interest  mean and SD of the fMRI time series, respectively. A variation of tSNR is the temporal contrast‐to‐noise ratio , while Zilverstand et al. , is referred to as the inefficacy problem , albeit mostly with fewer data to process. This means that rtfMRI algorithms can less likely detect true BOLD effects (or noise effects) as they occur, resulting in diminishing quality control of the rtfMRI‐NF signal.3.2.3Most scanner setups require custom modifications to hardware and/or software in order for extra physiological information to be transferred in real‐time. For example, to our knowledge few reports exist of physiological data (respiration and heart rate) being transferred and incorporated into a rtfMRI‐NF software pipeline to remove physiological noise in real‐time  are not critical for conventional fMRI analysis, they can cause substantial delays in real‐time processing and feedback presentation. However, specific details such as the tools and software versions used for data export and the real‐time latencies are rarely reported, which complicates reproducibility of methods. Additionally, differences in voxel intensity scaling, image orientation and image header information have been reported  reported the use of a standard list of real‐time pre‐processing steps. We conducted a Web of Science search across All Databases on April 9, 2019 using the same search terms and selection criteria as provided by Thibault et al. . Data and code necessary to reproduce Figures https://github.com/jsheunis/quality-and-denoising-in-rtfmri-nf), which also links to an interactive environment allowing exploration and visualisation of the study data.To facilitate further meta‐analyses and systematic reviews, studies should not only ensure a high level of data quality  but also have to consistently and comprehensively report their use of acquisition and processing methods. A further search of rtfMRI‐NF literature (including methods reviews) showed that rtfMRI‐NF processing methodology has been covered in some detail  if no mention of the particular method was made in the article or supplementary material, and if we could not confidently infer its use from studying the particular article's content. A possible exception to this rule is volume realignment, which could reasonably be expected to be used in almost all recent rtfMRI‐NF studies. Figure Figure Further results in Figure Finally, Thibault et al. noted a lack of both pre‐registration of rtfMRI‐NF study designs and registered report type publications, as well as a lack of adoption of general open science principles. Although open source software solutions like AFNI's real‐time plugin , bite bars, foam pads or end‐tidal forcing systems—to counter head motion or respiratory rate variation artefacts, respectively, but also extends to tweaking pulse sequence parameters or implementing alternative sequences to increase BOLD sensitivity. Several pulse sequences, hardware changes and other acquisition steps are highlighted below.As with conventional fMRI, it is recommended that researchers take the necessary precautions to mitigate the introduction of any unwanted noise sources into the data. This includes the possibility of using physical interventions—e.g. individualised head restraints  for these scanner effects.S0  and T2*  for denoising purposes is strongly recommended. While it may not always be possible to correct for physiological noise or motion in real‐time using these measures  they should at least be used offline to calculate and comment on correlations with BOLD fluctuations, task design or other subject‐related or experimental confounders.5.2As previously mentioned, real‐time denoising methods tend to follow the course of standard offline fMRI pre‐processing, although reports on the use of individual steps vary. Section processing methods since there would mostly be no differences between acquisition methods implemented to improve data quality for real‐time versus conventional fMRI. It should be noted, however, that some artefacts like electromagnetic spikes or shimming errors could be of greater importance for real‐time analysis compared to conventional fMRI analysis, since they could possibly be compensated for offline. This possibility does not exist for neurofeedback applications, hence extra care should be taken at the acquisition stage to try and avoid such artefacts. Table To aid the reader's understanding and guide future use of these methods, Table 5.2.1Geometric distortion effects, if addressed, are mostly accounted for using specialised acquisition methods as presented in the previous section, although correction through real‐time processing is possible. An example is the point spread function (PSF) mapping approach developed by Zaitsev, Hennig, and Speck  that is 5.2.2Slice timing correction interpolates the data of different 2D slices acquired at slightly different time points along the hemodynamic response, such that the resulting 3D image represents brain activity sampled at the same time point , few rtfMRI‐NF studies report its use , or FRIEND .As one of the major noise sources in fMRI, head motion received much attention during initial algorithm development in real‐time fMRI. Cox and Jesmanowicz  developeRegression of the six HMP time courses (and their framewise derivatives and/or squares derived by Volterra expansion) is a typical step used in conventional fMRI pre‐processing to correct for residual motion effects , although the latter option was proposed for future studies.Correcting for signal drift is a relatively standard step in real‐time fMRI and could form part of the real‐time GLM procedure, where a linear term and/or basis set of low frequency drift terms are included as regressors, acting as a high‐pass filter. An inherent correction for baseline drift is also executed in some percentage signal change neurofeedback paradigms during feedback signal calculation, due to the cumulative global mean being subtracted from the averaged ROI BOLD signal  is a method employed in conventional fMRI analysis to improve SNR . Filtering can also remove some physiology‐induced variance, with the modified Kalman filter by Koush et al. , 2017a bModel‐based approaches follow the work done by Glover et al. , Birn etTime synchronisation of peripheral recordings and fMRI data is a legitimate challenge to model‐based correction of breathing and heart rate variability artefacts in real‐time, unless the challenge is avoided altogether by using advanced processing power and full recalculation of all available data for every iteration, as was done by Misaki et al. . Some glLastly, we found no examples of studies investigating and comparing the efficacy of different real‐time physiological noise removal strategies or their effect on the neurofeedback signal in rtfMRI‐NF, although regarding offline correction, it has been suggested that motion or physiological fluctuations do not drive neurofeedback learning effects  has been a very effective tool in finding nuisance networks in resting state fMRI, which can be regressed out of the fMRI time series for effective denoising. Esposito et al.  collect real‐time cardiac and respiratory traces and analyse them after the neurofeedback session to investigate possible correlations with the task design or other BOLD fluctuations. Physiological traces can also be incorporated into offline physiological denoising (e.g. RETROICOR) when assessing the BOLD signal for neurofeedback‐induced changes over time  approaches show promise in reducing susceptibility induced geometric distortion and increasing BOLD sensitivity, and are recommended for future implementation.From a processing perspective, real‐time denoising pipelines showed high similarity to offline counterparts, although some trade‐offs are made because of the time limitation and the iterative nature of real‐time processing. The effects of inclusion or exclusion of specific denoising steps in the real‐time pipeline on the quality of the neurofeedback signal were found to be unexplored except for a single study  is always recommended when possible, either to be used for real‐time denoising or otherwise to rule out as confounds during offline analysis.7.2Quality control and best practices in rtfMRI‐NF is markedly unexplored and unreported compared to conventional fMRI, where initiatives like MRIQC . In this way, both published data and methods can be queried by multiple researchers, paving the way for reproducible methods, results and inferences.Moving towards a scenario where the hypothesised usefulness of rtfMRI‐NF in a clinical environment can be investigated and demonstrated transparently will require studies with reproducible methods and results. In light of this, we echo the recommendations made by Thibault et al.  regardinR. L. and M. B. are, respectively, employees of Philips Research and Philips Healthcare in The Netherlands. The other authors have declared that no further competing interests exist.Stephan Heunis, Rolf Lamerichs, Svitlana Zinger, Bert Aldenkamp and Marcel Breeuwer conceived the manuscript outline. Stephan Heunis conducted the research and wrote the manuscript. Rolf Lamerichs contributed to real‐time fMRI acquisition sections through discussions and writing. Cesar Caballero‐Gaudes and Jacobus F. A. Jansen contributed extensively during manuscripts revision periods. All authors reviewed and revised the manuscript.Data S1 Supplementary Notes.Click here for additional data file.


To generate evidence on willingness to use HIV self‐test kits and willingness to pay among antenatal care clients in public and private facilities in Cote d’Ivoire and Tanzania.Cross‐sectional survey data were collected from 414 clients recruited from 35 high‐volume facilities in Cote d’Ivoire and from 385 clients in 33 high‐volume facilities in Tanzania. Surveys covered willingness to use HIV self‐test kits, prices clients were willing to pay, advantages and disadvantages and views on specific qualities of HIV self‐tests. Market data on availability of proxy self‐testing products  and attitudes of pharmacists towards HIV self‐test kits were collected from 51 pharmacies in Cote d’Ivoire and 59 in Tanzania.Willingness to use HIV self‐test kits was 65% in Cote d’Ivoire and 69% in Tanzania. Median ideal prices women would pay ranged from USD 1.77 in Cote d’Ivoire to USD 0.87 in Tanzania. Proxy self‐test kits were available in pharmacies, and interest was high in stocking HIV self‐test kits.Implications for national HIV self‐test policy and planning include keeping prices low, providing psychological and HIV counselling, and ensuring linkage to HIV care and treatment services. Private pharmacies will play a key role in providing access to HIV self‐test kits. HIV testing is a prerequisite to accessing HIV care and treatment services, yet in some regions of sub‐Saharan Africa, only 48% of individuals living with HIV know their status . DispariRecent momentum towards country‐level regulatory approval and targeted distribution of HIV self‐test kits (HIVSTK) as an alternative to facility‐based HIV testing services offers a new and potentially important strategy to address testing gaps . HIV selIn 2016, the WHO published guidelines for HIVST , and manThis cross‐sectional study employed mixed methods to gather data on current markets and willingness to use HIVSTK and prices ANC clients were willing to pay for HIVSTK. Methods applied included mapping availability and pricing of self‐test proxy products. HIVSTK are not yet publically available in study countries, thus proxy products in the category of self‐test kits  were used to explore the provision of products similar to HIVSTK in the study areas. In addition, quantitative structured surveys were used to describe the willingness to use HIVSTK among a cross‐section of female consumers of ANC services and potential providers and suppliers, respectively.HIV self‐testing is new, with access most likely to be initiated in larger urban areas. For this reason, the study setting focused on four urban areas in each country. We selected Cote d’Ivoire and Tanzania as countries interested in piloting HIVSTK and at the time of the study were exploring ways to make the kits available. In Cote d’Ivoire, Abidjan and Bouaké districts, and in Tanzania, Dodoma and Arusha city districts were selected. In each study area, public and private health facilities and pharmacies were purposively selected for inclusion in the study. Private sector facilities included for‐profit facilities and faith‐based facilities; public sector facilities were government‐supported and managed facilities. Data were collected in 2018, from May 17th to June 9th in Tanzania and from June 4th to 22nd in Cote d’Ivoire.The Health Facility National Database was used to select facilities for client recruitment in Cote d’Ivoire. From a list of 255 health facilities (public and private), 35 facilities were identified as meeting the criterion of high volume and selected for client survey recruitment. In Tanzania, the Health Facility Register listed a total of 182 facilities, of which ANC clients were recruited from 33 high‐volume facilities.For the market assessment, pharmacy shops located near the selected ANC facilities were selected, comprising 51 public and private pharmacies in Cote d’Ivoire and 59 private pharmacies in Tanzania.The study population consisted of ANC clients and pharmacists. Convenience sampling was used as the most feasible option to facilitate rapid recruitment ANC clients for structured surveys during their routine clinic visits. Eligibility criteria for participation in the structured surveys included being a current ANC client in a private or public study facility and age 18 years or older. All participants provided written informed consent prior to interview. Pharmacists who offer HIV proxy products  were recruited during visits to purposively selected pharmacies located in the service areas of study facilities. A sample size estimate of 192 ANC clients per district, per country, was based on a conservative estimate of 50% of ANC clients being willing to use HIVSTK and allowed an estimate within a margin of error of 5% of the population proportion.All mapping and cross‐sectional survey data were collected electronically using handheld tablets by trained data collectors. All data collection forms were standardised across the study countries with range and value checks programmed into the data collection forms to reduce data entry errors. Measures of willingness to pay were adapted from the literature on stated preferences using what price the respondent considers normal or ideal, too expensive and too cheap. The survey began with a short description of an oral HIVSTK. Data collection forms were developed in English and translated into French and Swahili for data collection in Cote d’Ivoire and Tanzania, respectively.Analysis of market mapping and ANC client surveys was performed using Stata version 15.0. Descriptive statistics were used to summarise categorical  and continuous variables . Willingness to use an HIVSTK was estimated as the proportion of ANC clients surveyed who said they would ‘surely use’ an HIVSTK in the future. Logistic regression models (unadjusted and adjusted) were used to identify factors associated with willingness to use HIVSTK in the future. Tests of independence were conducted using chi square to compare key outcomes across districts. The potential for HIVSTK access and use was assessed by triangulating data from market mapping and structured surveys among ANC clients.National and US‐based institutional review boards (IRB) approved this protocol including the National Committee for Research Ethics in Cote d’Ivoire; the National Institute for Medical Research IRB in Tanzania, and the Advarra IRB in the United States.Results of assessing market availability of HIVST proxy products are outlined in Table Pharmacists had positive perceptions of HIVSTK. Nearly 91% of pharmacists in Cote d’Ivoire said they would be interested in stocking HIVSTK and 78% said the same in Tanzania (data not shown). Table A total of 799 ANC clients were surveyed; 414 in Cote d’Ivoire and 385 in Tanzania. Table P < 0.001). Socioeconomic status (SES) was measured using a household assets index. There were significant differences in SES with 64% vs 48% medium SES and 10% vs 29% low income in Cote d’Ivoire and Tanzania, respectively.There were notable differences between the two country samples; however, in education, source of income, and socioeconomic status. In Cote d’Ivoire, more than half of women had only primary (29%) or no education (23%), vs in Tanzania where most women had at least primary (40%) or secondary (44%) education and very few (3%) reported no formal schooling  ($1.73 USD) for any self‐test kit they used in the past. In Tanzania, 67% of women reported ever using a self‐test kit for pregnancy, less than 5% had used other types of self‐test kits such as for malaria or blood sugar. The median price paid in the past for any self‐test kit was 1000 Tanzania shillings (TSH) ($0.44 USD) and most paid less than 1500 TSH ($0.66 USD).In both Cote d’Ivoire and Tanzania, acceptability of HIVSTK was very high among all women Table . A totalP < 0.001). There was agreement on the importance of ease of use with nearly three‐quarters (71%) saying this was very important in both countries. As far as confidentiality of the test, a higher proportion of women in Cote d’Ivoire valued this more with 63% saying confidentiality was ‘very important’ vs (49% in Tanzania (P < 0.001). Attitudes were mostly positive towards using an HIVSTK in the future with at least 65% of women across the study countries saying they would sure use an HIVSTK in the future. However, more women in Tanzania said they would surely not use an HIVSTK in the future, (11%) that they disliked the idea of the HIVSTK and that they would feel very uncomfortable testing alone (22%) or sharing an HIVSTK with their partner (14%).Attitudes and preferences about HIVSTK were significantly different across Cote d’Ivoire and Tanzania. A higher proportion of women in Tanzania than in Cote d’Ivoire said accuracy of the test was very important, 71% vs 62%, respectively  women in Cote d’Ivoire and 146 (38%) women in Tanzania. In Cote d’Ivoire, other main disadvantages women mentioned were concerns about using the test correctly and getting a result that others would consider valid. For 178 (43%) women, the main disadvantage of HIVST was that the test result may not be accurate. In 174 42%) cases, the main disadvantage was that they may not know how to use the test kit, and for 41 (10%) that ‘nobody else would believe or trust the result’. In Tanzania, accuracy of the test and ease of using the test were the main disadvantages for only 39 (10%) and 42 (11%) of women. Few women said the main disadvantage is their partner becoming angry (4%) or someone else not believing the test result (3%) , and to the scant literature on prices, potential consumers are willing to pay for HIVSTK. Proxy products were readily available in pharmacies, and private sector interest in supplying HIVSTK was high.Pricing will be a crucial aspect of ensuring access to ordinary consumers. Women in our study were willing to pay an ideal median price of $1.77 USD in Cote d’Ivoire and $0.87 USD in Tanzania for an HIVSTK. Other studies on willingness to pay for HIVSTK, including in Tanzania, similarly demonstrate preferences for low‐cost HIVSTK , 17, 18.A small proportion of women in our study had health insurance, and although having insurance was a predictor for being willing to use an HIVSTK in the Cote d’Ivoire bivariate model, the generally low access to health insurance in both countries is likely to have little impact on uptake of HIVST. Nevertheless, promoting mandating that health insurance coverage include HIVSTK may be one component in a set of national strategies for increasing access.More economic resources or access to health insurance may facilitate health seeking from the private sector, but only if people perceive the private sector to be providing quality services/products. Our findings on the advantages and disadvantages of using the test kits described in this study highlight the practical, structural and cultural complexities that HIVST policies must consider in order to support high HIVST acceptance. Because the HIV testing infrastructure has been established as a free service, conducted by medical professionals, and under government guidelines, any new modality for HIV self‐testing through the private sector (and largely through pharmacies) will require shifting attitudes and building trust in the quality of those services. For example, one of the main disadvantages women in our study said was the lack of psychological and HIV counselling support if self‐testing. This may be addressed by providing support and links to care through a call‐in number or adding HIVST information and counselling support to existing hotline numbers.. In Nigeria, a call‐in number was provided to recipients of HIVSTK, half of participants who tested HIV positive called in for additional support . PrivacyPrivate pharmacies work in informal coordination with public facilities by providing convenient, though with a cost, access to medicines prescribed by clinicians at public facilities, where those medicines are often out of stock in that facility's pharmacy. In addition, they play a role in facilitating access to over‐the‐counter medicine, often purchased in lieu of a clinical/health facility visit where clients perceive advantages regarding opportunity costs , and they provide access to elective self‐care products such as condoms and health monitoring kits (e.g. high blood sugar). The majority of private pharmacy respondents reported interest in incorporating HIVSTK into their services. In Cote d’Ivoire, only 73% of pharmacists had ever heard of HIVSTK; however, acceptability of the approach was high, with over 90% reporting interest in stocking them in the future. In Tanzania, 56% of pharmacists knew about HIVSTK and 78% would want to stock them.The main challenges for pharmacists are similar to other essential commodities that are newly introduced and include stock outs, transport, supply and cost or pricing challenges. Engaging with the private sector could greatly expand the locations where HIVSTK are made available. This would require some investment in training private sector pharmacy staff in the reporting systems used in the public sector for HIV testing services, as well as ensuring adherence to other health systems regulatory requirements. Pharmacists will require training and guidance in offering HIVSTK, and potentially building pharmacy staff skills in providing pre‐ and post‐test counselling and links to confirmatory testing, care and treatment services, and/or establishing call‐in numbers available to access counselling or help troubleshoot testing challenges, as well as linking callers to care. Some pharmacies may lack the physical space for individuals to receive confidential counselling, and this should be a consideration in ensuring quality of services provided. Therefore, a coordinated health systems approach engaging public and private sectors to strengthen supply chains, establishing acceptable pricing, providing training for HIV counselling and testing and HIV reporting systems, guidance in offering HIVST, and coordinating linkages to care and follow‐up systems is required.There are several limitations to this study. HIVSTK are not yet available in Cote d’Ivoire or Tanzania, thus none of the study participants had actual experience with HIVSTK. Views on HIVSTK, advantages and disadvantages, important qualities and price were all provided in the hypothetical, which may not be an accurate predictor of future behaviour. This was mitigated by interviewers using a standardised script explaining what an oral HIVSTK is and how it is used.The study was not designed to assess male partners’ perspectives and views on HIVSTK, or if they would use an HIVSTK shared with them by their female partner. Some gendered differences exist on motivations for men in HIV testing in the context of couples , 24 and HIVSTK were found to be highly acceptable among ANC clients in Cote d’Ivoire and Tanzania, with the price that women are willing to pay for them similar to that for pregnancy self‐test kits. Providing HIVSTK at low cost will likely ensure more consumers will use them. Consideration should be given to providing psychological and counselling support, and ensuring linkage to HIV services for those testing positive. Engaging the private sector, including pharmacies, will be an important strategy to optimise implementation of an HIV self‐testing program.The governments of Cote d’Ivoire and Tanzania are currently exploring how to integrate HIVSTK into their policies. These findings will be useful in the formulation of other sub‐Saharan African national HIVSTK policies and to inform national planning for introduction and roll‐out in order to strategically maximise access to HIVSTK for high‐risk populations.


We exploit the possible link between structural surface roughness and difficulty of crystallisation. Polymorphs with smooth surfaces may nucleate and crystallise more readily than polymorphs with rough surfaces. The concept is applied to crystal structure prediction landscapes and reveals a promising complementary way of ranking putative crystal structures. The link between structural surface rugosity and ease of crystallisation has been explored (see picture). Smooth polymorphs were found to crystallise more readily than rough polymorphs. A code was written to compute crystal rugosity and applied to crystal structure prediction calculations, thus affording a new way of linking landscapes to experimentation. Perhaps one of the most enduring problems associated with the solid‐state chemistry of pharmaceutical materials remains the issue of polymorphism, where an active ingredient may adopt a number of different crystal structures.1, 2 TCSP has evolved tremendously in the last twenty years or so to the point where this technique is now routinely used in industry.4, 5 AAs a starting point we note that one central parameter common to both nucleation and growth processes is the interfacial tension. In nucleation theory this appears as a thermodynamic factor, which dictates the concentration of critical nuclei existing under certain conditions of supersaturation and temperature. In crystal growth it appears as the edge energy controlling the distance between spiral steps in dislocation‐controlled growth and the concentration of 2D nuclei in growth controlled by surface nucleation. The results of two recent studies are of relevance here. In the first it was reported that through neat grinding the stability of polymorphic forms could be reversedC* of nuclei would be greater for forms with lower values of interfacial tension so that nucleation is favoured for those forms which on balance have smooth surfaces. As far as growth of these nuclei is concerned, the dead zone review of Liu et al. shows that growth too is linked to roughness with rough surfaces potentially exhibiting zero growth at low supersaturation.The former of these observations is not unexpected since, given the Ostwald–Freundlich relationship between crystal size and solubility it follows that for two polymorphs there may indeed be a transition size at which the relative stability of forms switches.hkl) face (Rhkl)(depth) of a crystal, we calculate the degree of interpenetration between two consecutive crystal (hkl) layers along a particular crystal direction using the method of Bryant, Maloney and Sykes.Rhkl) crystal slice and the average plane between that slice and its consecutive slice. We note that the sign of the rugosity parameter simply refers to whether the surfaces are interpenetrated (negative) or separated (positive) by the value Rhkl)(depth are discussed in absolute terms (for example a surface with a Rhkl)(depth>0 Å is considered perfectly smooth). We have written a python program using the Cambridge Structural Database (CSD)hkl) values for its dominant faces,[d‐spacings (dhkl), the morphological importance for those faces (whkl) and their minimum rugosity value (Rmin-depthhklin Å). The minimum rugosity value is assessed from four independent calculations of rugosity for slices created at the same (hkl) value with their origins shifted by various fractions of dhkl) plane, the lowest value of all computed Rhkl). An overall normalised crystal rugosity value is then defined for a given crystal as the sum of the minimum rugosities of each (hkl) face normalised by the face d‐spacing and weighted by its BFDH morphological importance. The normalised crystal rugosity R‾depthNis then calculated as:To quantify the surface rugosity of each (Rmin‐depthhkl). The distribution of such difference is given in Figure ΔR‾depthNdiffer by a maximum of 0.08, 96 % by 0.17 and over 98 % by a maximum of 0.20. This histogram reveals the maximum difference in normalised crystal rugosities within polymorphic families in the CSD and these statistics have the potential to be used for interpreting CSP landscapes by limiting the “realisable forms” to those with lowest rugosities . To test the utility of this conclusion and its application to polymorphic materials we have constructed a dataset of polymorphic pairs which have been reported to be either easy or difficult to crystallise , the form with the rougher surfaces is usually also the form which is harder to crystallise (thus ΔR‾depthNehis positive). In examining the link between rugosity and polymorph stability it is noted that these are not necessarily correlated. However, when the rougher crystal form is also the most stable, thermodynamics favours this form but kinetics does not. These are the cases of theophylline‐IVThe attribution of a given polymorph to the category “difficult” is made on the basis of it not being obtainable from straightforward solution crystallisation. This definition includes polymorphic forms that can only be obtained by solid‐solid transformations, solvent‐mediated transformations or by desolvation as well as conditions which require the presence of impurities or high pressure (see ESI). By contrast, a polymorph is noted as “easy” if it is the most commonly found form under most crystallisation conditions. Here, we calculate the difference in normalised rugosities between the polymorph that is easy to crystallise and the polymorph that is hard to crystallise, R‾depthN=-0.057) and R (R‾depthN=-0.102) forms were smoother than the stable room temperature Y form (R‾depthN=-0.265).R‾depthN=-0.081) crystallises preferentially from aqueous solutions, while the stable γ (R‾depthN=-0.087) can be obtained only by using tailor‐made additives or working at a pH different from the isoelectric point. β‐Glycine has the lowest rugosity (R‾depthN=-0.039) and it quickly transforms to α but there is no information as to how often it nucleates before α.Next, we looked at a number of notorious polymorphic systems with many known forms (ESI). When experimental data on the stability of forms was available  of each crystal structure versus its normalised average crystal rugosity. The vertical lines bracket the 98 % attainable space between the smoothest polymorph in the landscape within 4 kJ mol−1 of the global minimum  and the rougher polymorph possible with −0.2 rugosity from the smoothest form . This idea flows naturally from our previous work on nucleation and growth of benzoic acids which demonstrated the link between nucleation and growth rates.ΔR‾depthNas a measure of crystallisability combined with the computed lattice energies and the fact that in most polymorphic systems ΔR‾depthNdoes not exceed 0.2, it is possible to identify those forms most readily accessible through experimentation. We do not expect this model to be infallible for several reasons: i) conformational aspects of crystallisation are important and also need accounting for, ii) some rough surfaces may be stabilised in solution due to favourable solvent–surface interactions, iii) the presence of small levels of impurities in the solution together with other more complex nucleation pathways may play a role in the observation of forms and iv) the method presented here is based on a simplified 1D description of rugosity and BFDH morphologies which make no account for solvent effects. Whilst we are now working to improve our method of crystal rugosity calculations, this novel concept and the method presented here can already be applied as a quantifiable tool bringing a new interpretation of CSP lattice energy versus crystallisability landscapes. Given how ubiquitous these landscapes have become in solid form development of pharmaceuticals, this method should be of interest to the community.In conclusion, we have derived a model to compute normalised crystal surface rugosities in molecular crystals based on the roughness model of Bryant, Maloney and Sykes.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


CMPR provided clear visualization of the classic “interlocking C” appearance of the dentate gyrus and cornu ammonis along the entire caudal–rostral axis including the head and tail, which otherwise show complex anatomy on the standard coronal slices. CMPR facilitated visualization of hippocampal anatomy providing the impetus to develop simplified approaches to delineate subfields along the entire hippocampus including the usually neglected head and tail.There is a growing body of literature studying changes in hippocampal subfields in a variety of different neurological conditions, but this work has mainly focused on the hippocampal body given challenges in visualization of hippocampal anatomy in the head and tail when sectioned in the typical coronal image plane. Curved multiplanar reformatting (CMPR) is an image reconstruction method that can improve visualization of complex three‐dimensional structures. The objective of this study was to determine whether CMPR could facilitate visualization of the human hippocampal anatomy along the entire caudal–rostral axis. CMPR was applied to high‐resolution magnetic resonance imaging acquired ex vivo on four cadaveric hippocampal specimens at 4.7 T (T2‐weighted, 0.2 × 0.2 × 0.5 mm In ordeThree‐dimensional curved multiplanar reformatting (CMPR) has been used to improve visualization of tubular structures with complex three‐dimensional orientation such as blood vessels and the tracheobronchial tree . As expected, there was marked signal attenuation at the ends of the short length RF coil. While it was possible to scan Hippocampus 1 completely, only the hippocampal head and body were successfully imaged for the remaining three hippocampi.Four hippocampi from different individuals with no history of neurological disease were obtained postmortem . Specimens were fixed in formaldehyde for an average of 6 months, trimmed to fit into a 50 ml centrifuge tube and immersed in a liquid fluorocarbon . Specimens were scanned at room temperature in a 4.7 T MRI  using a custom‐built volume radiofrequency (RF) coil  and a T2‐weighted (inverted contrast) fast‐spin echo technique with 80 contiguous 0.5 mm coronal slices perpendicular to the long axis of the body of the hippocampus . The selection of the plane of view was performed manually in order to maintain a transverse orientation to the long axis of the curved hippocampus  , dentate gyrus, and cornu ammonis is clearly visualized with ex vivo imaging Figure . HippocaWhile tremendous advances have been made regarding the segmentation of the hippocampal body  of resliced sections of the posterior hippocampus suggesting that CMPR can facilitate visualization of the hippocampal tail (Adler et al., While we have successfully applied the CMPR method of visualizing hippocampal internal architecture on very high resolution T2‐weighted MRI in four ex vivo hippocampal specimens, a limitation of this technique is that the CMPR approach was manual, making it susceptible to user bias and being labor intensive. The sharp anterior curve of the arc of the hippocampus anteriorly, could result in variability in the selection of the orientation of the imaging plane between users and it will therefore be necessary to determine inter and intra rater reliability in particular if CMPR is used to measure hippocampal subfield volumes. While the application of our previously reported methods for segmenting the hippocampal body to the CMPR data demonstrates feasibility, we were unable to confirm the accuracy of the labeling as our specimens were sectioned orthogonal to the A‐P axis. Despite this limitation, our preliminary results are encouraging and suggest that CMPR could be applied to ex vivo MRI, histology, and potentially in vivo MRI in order to facilitate visualization and measurement of hippocampal subfields along the entire length of the hippocampal axis.The authors declare that they have no conflict of interest.2Video S1 Curved multiplanar reformatting demonstrates that the “interlocking C” relationship between the cornu ammonis and dentate gyrus is maintained throughout the entire length of Hippocampus 1.Click here for additional data file.


Flowers have been hypothesized to contain either modules of attraction and reproduction, functional modules (pollination‐effecting parts) or developmental modules (organ‐specific). Do pollination specialization and syndromes influence floral modularity?Erica: we gathered 3D data from flowers of 19 species with diverse syndromes via computed tomography, and for the first time tested the above‐mentioned hypotheses via 3D geometric morphometrics. To provide an evolutionary framework for our results, we tested the evolutionary mode of floral shape, size and integration under the syndromes regime, and – for the first time – reconstructed the high‐dimensional floral shape of their most recent common ancestor.In order to test these hypotheses and answer this question, we focused on the genus We demonstrate that the modularity of the 3D shape of generalist flowers depends on development and that of specialists is linked to function: modules of pollen deposition and receipt in bird syndrome, and access‐restriction to the floral reward in long‐proboscid fly syndrome. Only size and shape principal component 1 showed multiple‐optima selection, suggesting that they were co‐opted during evolution to adapt flowers to novel pollinators. Whole floral shape followed an Ornstein–Uhlenbeck (selection‐driven) evolutionary model, and differentiated relatively late.Flower shape modularity thus crucially depends on pollinator specialization and syndrome. In genet al., et al., et al., et al., et al., et al., et al., et al., et al., all the distances between all the 30 points (i.e. 435 distances). This property makes GM well‐suited to digitizing the complex 3D geometry of flowers and ideally suited for the study of modularity, which focuses on which feature is dependent or independent of which other. The few studies that used GM all were focused on integration and based on 2D pictures. Two‐dimensional pictures not only add noise to the digitization of data  with different pollination syndromes . We used phylogenetic reconstructions to test if floral parameters  evolved under selection driven by pollination syndromes or randomly. We thus aimed to understand: (1) The relative utility of the different components of floral shape (i.e. which Cartesian coordinate of which landmark) and size in predicting pollination syndromes; (2) how floral shape modularity changes with pollination syndromes and floral specialization; (3) the possible evolutionary patterns of floral shape in Erica; and (4) which of the natural selection models (i.e. Ornstein–Uhlenbeck) and drift‐like models (i.e. Brownian motion) best explain the evolution of floral shape, size and integration with respect to pollination syndromes.In order to test the above‐mentioned modularity and macro‐evolutionary hypotheses, we generated 3D models of c. 10 flowers each from a single genotype representing nineteen species of Erica from the collections of the Belvedere Garden . We selected one genotype per species because it afforded us the most reproducible sampling of floral variation; moreover, in different systems, most floral trait variance has been shown to lie within individuals   software suite. Geometric morphometric landmarking was carried out on surface models in Amira (generated via the isosurface function). Thirty‐three homologous landmarks were placed on each flower   and one loci of the nuclear genome ) from 61 pre‐existing sequences of the 19 Erica species as ingroup, and Calluna vulgaris and Daboecia cantabrica as outgroups   for the 19 Erica species to estimate the fit of three different evolutionary models; Brownian Motion , Ornstein–Uhlenbeck  and Early Burst  in order to better understand the process of floral‐shape evolution in the clade     was warped (distorted) to each target ancestral shape. The warping was carried out by aligning the reconstructed ancestral shape at the selected nodes and the landmark data of the chosen model (E. hirtiflora) using a thin plate spline (TPS) interpolation  , we fitted a series of likelihood models: BM1  and BMS ; and OU1 , OUM (selection‐like model with a single rate but different optima for each group of taxa) and OUMV (selection‐like model with different optima and different rates for each group of taxa). The best‐fitting model was determined comparing AICc and AICc weights among the models. All analyses were implemented using R/ouwie . The convergent evolution of the bird and LPF syndromes in our dataset displays different patterns: the LPF syndrome flowers are tightly clustered in the morphospace whereas the bird syndrome flowers are in two clusters.Together, principal component (PC) 1 and PC2 account for 62% of total shape variation (38.9% for PC1 and 22.1% for PC2). The main distortion along the PC1 is a constriction, elongation and slight curving of the corolla tube. Flowers along PC2 are differentiated mainly by the proximal to medial position of the inflation of the corolla. This varies from globose‐urceolate to tubular‐urceolate flowers along PC1 and cylindrical to ovoid floral shape along PC2. The PC axis‐related distortion along PC1 and PC2 is visualized by an exemplary shape distortion of a flower of developmental hypothesis  and 2 (restriction) received weaker support  will be detailed here (i.e. LPF and wind syndromes). In the flowers with LPF syndrome, large flowers tend to have a more flask‐shaped corolla, and the landmarks on the mouth of the corolla are closer to the floral axis .Under the penalized likelihood approach, the best‐fitting model to the evolution of the highly‐dimensional whole floral shape in ers Fig. b, node 11, BMS, OU1, OUM and OUMV) on quantitative floral trait evolution  under the four pollination‐syndrome regimes are summarized in Table MV) displayed a negative eigenvalue for PC3, PC4, integration and centroid size, which means that this model was too complex for the information contained in these data and it was excluded from the analyses. Different evolutionary models yielded variable AICc distributions and AICc weights , and invalidates both the ‘trait specialisation’ and ‘common shape’ hypotheses . Our findings contrast with most published studies that recover support for the functional hypothesis in flowers with generalist syndrome  have been reported widely  which display support for (different) Both the functional hypothesis 1 (pollen deposition/receipt) and the developmental hypothesis are well supported in bird‐pollinated flowers; this supports the hypothesis that developmental modularity is a default modularity, a form of null hypothesis Herrera, . The higErica flowers with this syndrome, however, do not always have narrow tubes, but do have a narrow corolla apertures  and to vary much less than the rest of the flower . The strong but weakly significant allometry reflects typical differences in flower shape related to differences in anthesis stage: larger (older) flowers have more open petals and more exerted stamens than smaller (younger) flowers  wind‐pollinated flowers probably do not require modules across organ classes (for pollen receipt), and (2) that our data are dominated by developmental shape changes. Modelling studies in grasses that have shown that pollen deposition overwhelmingly relies on direct impact on the stigma and not on air flows generated by the rest of the flower  and showed an overall recent slowing down of speciation rates , the variable we demonstrate to play the strongest role in predicting the different syndromes  or a random model (Brownian Motion (BM)1) of evolution  together with geometric morphometrics to uncover the modularity of the highly dimensional shape of flowers as a function of pollinator syndrome, and together with a novel penalized likelihood framework  should be directed to the Fig. S1 Machine learning.Fig. S2 Hypotheses test: RV distributions.Fig. S3 Allometry in Erica flowers.Click here for additional data file.Methods S1 Details of the methodologies used to for: X‐ray tomography, 3D‐landmarking, geometric morphometrics, pollination syndrome prediction, modularity analyses (exploratory and confirmatory approaches), phylogenetic inference, ancestral character states reconstruction and models of trait evolution.Notes S1 Literature analysis.Notes S2 Pollination observation Erica gracillis in cultivation, allometric regressions, and correlation between the corolla tube length and centroid size.Click here for additional data file.Table S1 Species, sample numbers (n) and scanning conditions of Erica flowers.Table S2 Landmarks used to digitize the shape of Erica flowers, and modules to which they belong in the modularity hypotheses tested.Table S3 Species‐level average values for size and integration.Table S4 Genbank accession numbers for nrDNA ITS and cpDNA trnL‐F‐ndhJ and trnT‐L sequence data.Table S5 Discrete character‐mapping models for pollination syndromes.Table S6 Main variables mean accuracy decrease of RF syndrome prediction.Table S7 Corolla tube length per flower.Table S8 Classification of 114 individual flowers of diverse Erica species into the pollination syndromes.Table S9 Support values for evolutionary models of floral shape evolution.Table S10 Summary of the preferred models of evolution for seven phenotypic trait variables (PC1‐5 of floral shape, centroid size and integration.Click here for additional data file.


Horseshoe kidneys are found in about 0.25% of the population. They are commonly associated with other abnormalities like ureteropelvic junction obstruction (UPJO) and vescicoureteral reflux. UPJO can be caused by anomalous renal blood supply, high‐inserting ureter into the renal pelvis or its abnormal course over the isthmus, and intrinsic factors.et al. described the mini‐invasive surgical treatment of UPJO in five patients with horseshoe kidneys.Lu et al.Standard techniques suggest isthmusectomy in order to obtain a more physiological urine drainage. Despite this, Shadpour et al.There have been continuous debate in literature about the use of a retroperitoneal or transperitoneal approaches for the management of UPJO. A recent meta‐analysis concluded that both approaches are associated with high success and low complication rates, but transperitoneal pyeloplasty provides a shorter procedure time and lower conversion rate.The robotic approach has been documented in only seven cases in the literature.Multicentric randomized controlled trials with a longer follow‐up period are needed to establish an optimal treatment technique for this rare disorder.The author declares no conflict of interest.