Ascaris IgE, Ascaris eggs and Trichuris eggs) either in childhood or in adulthood, and residence in rural and resource‐limited urban areas influence allergy outcomes  in a South African population. Participants historical and present allergies data were collected through questionnaires and clinical record files. Coproscopy and immunoassays  were used for active helminthiasis and allergy screens respectively. Data were analysed using logistic regression analysis, and models were adjusted for age, gender and locality. High Ascaris IgE was significantly associated with asthma , IgE atopy  and food allergy . Asthma was significantly less likely among participants with Ascaris eggs  and Trichuris eggs . The findings of co‐occurrent helminthiasis and allergic disorders in a population that has resided both in rural and peri‐urban informal settlements both oppose and agree with two main notions of the hygiene hypothesis that (i) individuals residing in rural settings with poor sanitation and geohelminth infection are less prone to allergy, and (ii) helminth infections protect against allergy respectively. Further research is warranted.This study investigated whether prior exposure to helminths ( Permission to conduct the study was provided by the Matthew Goniwe Clinic management team. This cross‐sectional analysis used data obtained from the prospective study that were tracking the immune profile of adults during helminthic infection and anthelmintic treatment (n = 218), partly described in detail elsewhere.2.2Ascaris lumbricoides and Trichuris trichuria are the most predominant intestinal helminths in the Western Cape Province and Cape TownStudy participants were Xhosa adults (18 years and above) who were clinic attendees, presenting for different ailments, some were accompanying their relatives to the clinic and others were attending the clinic's HIV/AIDS support group. This is a Health District clinic of the Metro Region and is the largest in this region, servicing the informal settlement. These individuals were recruited by convenience sampling and participated in a larger prospective immunology and deworming study published in part elsewhere.2.32.3.1Lifetime history and current experience of asthma and rhinitis symptoms and treatment as well as current use of asthma inhalers and rhinitis nasal sprays or other treatment were collected through questionnaires administered in the local isiXhosa language. Clinical records were also used to confirm clinician‐diagnosed allergies and treatment where available. Asthma and rhinitis recall and clinical diagnosis were also recorded based on the use of prescribed medications including inhalers such as Ventolin and its generics , nasal sprays such as Beclomethasone (Beconase) nasal spray and its generics and other rhinitis prescriptions also recorded from patient files to confirm asthma and rhinitis, respectively, where data were available in case record files, as well as those carrying these medications to the clinic at the time of interviewing.A. lumbricoides and a few others such as T. trichuria and tapeworm species. These data were elaborated in detail in a previous publicationSimilarly, history of worm infection, as described in a previous study that enquired about history of intestinal helminth infection, more than 70% reported a recall of worm infection either as a child or an adult, more often both as a child or adult.2.3.2A. lumbricoides and T. trichuria are the most common helminths in the study area and were thus the focus of this study. Two stool samples, collected on two different days, were used for detection of helminth infection. Coproscopy, using Kato‐Katz and the modified formol‐ether concentration (Mini‐Parasep) by two independent microscopists, were used to detect the presence of helminth eggs. Mebendazole and praziquantel for schistosomiasis treatment were provided for all participants found to be infected with worms.Both A. lumbricoides‐specific IgE levels were thus analysed. Antibody cross reactivity with T. trichuria has been documented,Ascaris IgE is used  in the context of describing exposure/non‐exposure in order to classify those who had past or present helminthiasis infection. This analyte may be either a confounder or an intermediate factor in the association between helminth infection and allergy.Serology was also used to increase the sensitivity of microscopy for intestinal helminth infection or exposure. 2.3.3A. lumbricoides and/or T. trichiuria egg excretion plus serum A. lumbricoides ‐specific IgE (used as a proxy to indicate helminth exposure) as well as demographic variables such as age, gender and locality type (place of birth). The study biological outcomes included eosinophil counts, serum total IgE, total IgE, defined as overall quantity of all immunoglobulin E subtypes in blood. High total IgE for the study methodology are levels equal to or above the defined cut‐off value of 70 kU/L. Allergic disorder outcome variables included four measures: (1) IgE atopy , (2) asthma, (3) rhinitis and (4) food allergy ; mixed trees ; mixed weeds ; Bermuda and Timothy grass and mixed mould . Total serum IgE, food sensitivity and A. lumbricoides‐specific IgE levels were determined by ImmunoCAP Total IgE, ImmunoCAP allergin‐specific fx3 IgE, and Ascaris IgE radioallergosorbent (RAST) tests respectively. Food allergens included egg whites, milk, soya, peanuts, wheat and fish (cod and shrimp).The primary exposure variables were faecal easures:  IgE atop2.3.4A. lumbricoides‐specific IgE, stool A. lumbricoides and T. trichuria eggs excretion; as well as demographic variables such as age, gender and locality type (place of birth) . Exposure variables included helminth parasitic infection exposure variables: serum h) Table .2.4p‐value ≤.05 were used to determine the level of statistical significance. Data were analysed using STATA version 15.0 (Stata Corporation).Descriptive statistics frequencies and percentages were used to summarize the data. Pearson chi‐square test was used to test for differences between categorical variables. Bivariate and multivariate logistic regressions were used to analyse the association between measures of allergy‐related disorders and helminth parasitic infection exposure. Crude (OR) and adjusted odds ratios (aOR)) and 95% confidence intervals (95% CI) with a 33.1Ascaris (24.1%) and Trichuris (24.8%) eggs in stool and 33.9% had high Ascaris IgE. The overall prevalence of helminth infection was 43%, by egg excretion positivity; however, the prevalence as measured by either high Ascaris‐specific IgE, and/or Trichuris/Ascaris egg excretion was 66% in this sample population. In terms of allergic disorders, overall, asthma, rhinitis, IgE atopy and food allergy prevalence were 17.5%, 54.7%, 39.9% and 11.5% respectively (Table Table Ascaris 4.1% and Ascaris IgE: χ2p = .002 and Trichuris eggs: χ2p < .0001) and (ii) allergy‐related disorder outcomes  were observed. Those born in Cape Town had the highest prevalence of high Ascaris IgE (66.7%) and Trichuris eggs (83.3%) while Ascaris eggs were most prevalent in participants originally from the Eastern Cape (26%). Asthma prevalence was similar across all the places of birth and ranged between 16% and 18%. The Eastern Cape had the highest prevalence of rhinitis (55.6%) while IgE atopy (72.2%) and food allergy (33.3%) was most prevalent among those born in Cape Town (Tables The majority of participants (88.5%) had migrated from rural villages in the Eastern Cape (their place of birth) to the Western Cape Province's City of Cape Town informal settlement. Tables is eggs 8.3% whilen Tables  and S2.3.2Ascaris IgE. Rhinitis prevalence was similar and higher in participants that had active worm infection as shown by stool positivity for Ascaris eggs (65.3%) and Trichuris eggs (62.5%). All helminth exposed groups (high Ascaris IgE and positive stool Ascaris and Trichuris eggs) had a similar prevalence of asthma ranging between 24.3%, 24.5 and 25% respectively , rhinitis 51.4%) and food allergy (27%) were most prevalent among the group with high .4% and f3.3p < .001), IgE atopy (p < .001) and food allergy (p < .001). The biological reactants, eosinophilia and high total serum IgE are characteristic biological markers associated with both exposure variables (parasitic infections) and outcome variables . As expected, participants with high total IgE levels significantly associated with allergy‐related disorders . Likewise, high eosinophil levels were associated with asthma: p < .001, rhinitis: p = .012, IgE atopy: p = .001 and food allergy: p = .024  and those with Trichuris eggs  but more likely among those with high Ascaris IgE . IgE atopy was significantly more likely among those with high Ascaris IgE . Food allergy was also significantly more likely among those with high Ascaris IgE .Table 4A. lumbricoides  and egg excretion on allergic disorders including atopy, asthma, food allergy and rhinitis in participants from a socioeconomically deprived rural population that had moved to urban informal areas.Ascaris‐specific IgE. More than 20% had active worm infection shown by Ascaris (24.1%) and Trichuris (24.6%) eggs in stool and approximately a third (33.9%) had high Ascaris‐specific IgE. Overall occurrence of allergic disorders in the study sample ranged between 11.5%  and 39.9% (atopy), as measured by allergen specific IgE screen; and asthma (18%) and 55% rhinitis according to recall of disease symptoms and treatment. The study population generally had high serum total IgE. This is expected since helminth infections induce a polyclonal stimulation of IgE synthesis via IL‐4,Ascaris‐specific IgE, there could be cross‐reactivity.This study investigated the effects of exposure to Ascaris IgE also had high IgE atopy, and more than 50% had rhinitis. Those with active worm infection had more than 60% rhinitis, and all helminth exposed groups had more than 20% prevalence of asthma. In the present study, high Ascaris IgE showed a significant association with atopy and food allergy, both of which were increased after adjustment for demographic characteristics. Likewise, high Ascaris IgE was also significantly associated with asthma but significantly less likely among those with Ascaris and Trichuris eggs in stool (active worm infection). These results present high prevalence of both helminth infection and allergic disorders, and concurrent manifestation of both, in a population that has moved from rural villages into an urban informal settlement, with a history of childhood and/or adult worm infection and current evidence of worm infection. These findings do not support the hypothesis of an inverse relationship between parasite infection and allergic sensitization.Regarding the concurrent manifestation of allergic disorders and helminth exposure, the majority (78.4%) of those with high Ascaris‐specific IgE were fourteen times more likely to be atopic. This association was highly statistically significant. The significant association between atopy and high Ascaris IgE is a typical reflection of the documented presence of elevated Ascaris IgE being a risk factor for atopy or a genetic tendency towards allergic sensitization and asthma or wheezing.Contrary to previous studies that have suggested that helminth parasites protect against atopy,Ascaris infection. In addition, active worm infection (Ascaris and Trichuris eggs in stool) was also significantly associated with males, although they only constituted a smaller proportion (14%) of the study sample. This is in keeping with the general finding that males are generally more susceptible to parasite infections.Other findings showed that male gender was associated with The findings of the current study also add another layer of ambiguity to the rephrased hygiene hypothesis that incorporates the acquisition of ‘old friends’ such as helminths, and microbiota early in life as primers of immunoregulation that results in an appropriately responsive immune response later in life.Ascaris IgE were significantly more likely to have asthma, food allergy and atopy, opposing the first point. Nonetheless, those with active worm infection were found to be significantly less likely to have asthma, which is in agreement with the second point. Some epidemiological reports suggest that allergy risk is reduced in high geohelminth infection settings as is the case with the current study area.These findings both oppose and agree with two main notions of the hygiene hypothesis that (i) individuals residing in rural settings with poor sanitation and geohelminth infection are less prone to allergyThere are contradictory reports on the occurrence of allergic diseases in low‐ and middle‐income countries; however, urban areas are reported to be most affected than rural areas.Ascaris, due to their conspicuous size, egg excretion and Ascaris‐specific IgE were also measured and confirmed high prevalence of the two helminth species. Likewise, for the history of asthma and rhinitis symptoms and treatment, current symptomatology and treatment were also recorded and confirmed through clinic records to mitigate the recall and self‐reporting bias. In addition, for allergic disorders, blood measurements for IgE atopy and food allergy as well as total and Ascaris‐specific IgE were undertaken. The study was also limited by the small sample size which is reflected by the wide confidence intervals in some of the associations. An additional confounder was gender bias since the majority of participants were females. This was a default representative of a healthcare sample, as is a common phenomenon where females are more likely to be healthcare seekers than males. Demographically, most participants were born in the Eastern Cape villages.However, the study has several limitations. Firstly, it is limited by an interviewer‐administered, retrospective questionnaire which may have possible recall bias for asthma and rhinitis. However, there were several steps in mitigating some of these limitations. For example, in addition to the fact that for the history of worm infection, the worms described mostly resembled Ascaris egg excretion and asthma. During the early phase of Ascaris infection, the larvae migrate to the lungs before being coughed up, causing an eosinophilic pneumonia‐like symptom that may present as asthmaA spurious finding was the significant negative association between Although limited by several factors, the study highlights that early and current infection with helminths and exposure to rural and later, unhygienic environments  do not preclude the occurrence of allergies induced by food and inhalant allergens, and asthma and rhinitis.5We have shown more than 20% concurrent active worm infection and allergy in adults with evidence of lifelong exposure to environmental stimulation factors  as well as helminthiasis. This finding seems to be in agreement with the proposition that complex factors interact to determine the occurrence of allergic and autoimmune diseases in the context of helminth infections. This report adds to the ever‐contradictory studies of the interactions between helminths and hyperimmune disorders. More insights into the extension of the reformulated hygiene hypothesis and old friends’ theory are neededThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Zilungile Lynette Mkhize‐Kwitshana contributed to conceptualization, laboratory and field work and writing—original draft preparation. Pragalathan Naidoo contributed to conceptualization and writing—reviewing and editing. Ntombifikile M. Nkwanyana contributed to data analysis and validation. Musawenkosi L. H. Mabaso contributed to conceptualization, data analysis and writing—reviewing and editing.Fig S1‐S2Click here for additional data file.


Despite decades of effort, predicting the El Niño-Southern Oscillation (ENSO) since the 2000s has become increasingly challenging. This is due to the weaker coupling between the ENSO and well-known precursors in tropical ocean basins, particularly in the Indian Ocean. Here we show that the Southern Indian Ocean Dipole (SIOD), which is characterized by an east-west-oriented sea surface temperature dipole pattern over the southern Indian Ocean, has become a key precursor of Central Pacific El Niño since the 2000s with a 14-month lead. The role of the SIOD in the subsequent year’s ENSO is distinctive from the equatorial Indian Ocean Dipole mode in that it prolongs the ENSO period. The westward-shifted ENSO has sustained simultaneous SIOD events for longer periods since the 2000s, which leads to weak but persistent westerly anomalies over the western Pacific. This eventually results in the development of the Central Pacific El Niño in the subsequent year. Predicting the El Niño-Southern Oscillation (ENSO) since the 2000s has become increasingly challenging. Here the authors show that the Southern Indian Ocean Dipole has become a key precursor of Central Pacific El Niño since the 2000s with a 14-month lead. The physical mechanisms underlying the evolution of El Niño and La Niña events and their transitions are reasonably well understood. The occurrence of El Niño events is led by the accumulation of the equatorial warm water volume (WWV) as a result of the slow ocean adjustment process during La Niña events, indicating a lead−lag relationship between the WWV and the ENSO6. However, this relationship has weakened since the 2000s7, suggesting that the WWV-related recharge/discharge process driving the transition of the ENSO phase has become less efficient, thus ENSO phase transitions have become less regular compared to the 1979–1999 period. In addition, ENSO behaviors have become increasingly diverse, thus it has become more difficult to predict ENSO events since the 2000s10.The El Niño-Southern Oscillation (ENSO) irregularly fluctuates between El Niño and La Niña conditions in the tropical Pacific with massive socio-economic and environmental impacts worldwide7. This is evident for the dominant sea surface temperature (SST) variability in the Indian Ocean (IO), which is known to lead the ENSO in the subsequent year14. Since the early 1990s, the influence of the Indian Ocean Dipole (IOD) and the Indian Ocean Basin-wide mode (IOBM) on the subsequent year’s ENSO has significantly weakened15 , which is characterized by an east-west-oriented SST dipole pattern over the southern IO, could be a powerful precursor to Central Pacific (CP) El Niño events occurring after the 2000s.The non-stationary relationship between the ENSO and large-scale oceanic variabilities outside the tropical Pacific can also hinder the successful forecasting of the ENSOd15 Fig. . On the 21 which appears as a 2nd EOF during the 1976–1997 period  season during the 1998–2019 period as a 2nd empirical orthogonal function (EOF) of the SST anomalies over the IO Fig. , differiiod Fig. . The SIO IO Fig. . Based o IO Fig. . Note th IO Fig.  and the  24 Fig. , the phy 24 Fig.  and S6.F25. After 15 months, a neutral state is present over the equatorial central-eastern Pacific26  are also observed.In terms of the SIOD during the OND season from 1998–2019, the spatial distribution of SST anomalies is similar to the positive IOD with a southward shift Fig. . As withIntriguingly, the SIOD-induced SST anomalies over the equatorial Pacific in the winter of the subsequent year completely differ from those induced by the IOD. After 14 months, positive SIOD events induce El Niño events, which is significant at a 95% confidence level Fig. . In part29 or Ningaloo Niño/Niña23, which are characterized by cold/warm SST anomalies during the boreal winter or early spring season in the southeast IO off Australia. The SEIO acts as a precursor of the ENSO for 9 months28, which is shorter by 5 months than that of the SIOD. Therefore, the correlation coefficient between the SEIO during the OND season and Niño3.4 during the December-January-February (DJF) season in the subsequent year is only −0.25  for 1998−2019. Similarly, the influence of Ningaloo Niño/Niña during the OND season on the subsequent year’s ENSO is negligible at the 95% confidence level  modevel Fig. . The strvel Fig. .31. It should be noted that the auto-correlation of Niño3 and Niño4 gradually decays during the following year and eventually transits into a neutral state in the winter of the subsequent year  shows two positive peaks at a lag of zero , where (0) denotes the simultaneous year for the SIOD peak phase) and 13 months J(+2), where (+1), and (+2) denote one, and two years after the SIOD peak year, respectively)  anomalies over the western-central Pacific during the following year’s boreal summer by raising the sea surface height and deepening the thermocline depth along the equator Fig. . As the ter Fig. .34. The weaker amplitude of SIOD-related thermocline feedback might be caused by the occurrence of simultaneous El Niño during the positive SIOD, leading to a discharge of equatorial heat via poleward geostrophic currents35. As the subsurface temperature anomalies over the equatorial Pacific have negative values due to heat discharge, neglectable positive vertical temperature advection is induced. Additionally, the SIOD-related westerly anomalies over the equatorial western Pacific are persistent but relatively weak in terms of their amplitude compared to that of conventional El Niño events  reflect a weak contribution from thermocline feedback  and a significantly stronger contribution from zonal advective feedback  over the equatorial western-central Pacific when compared with El Niño events without SIOD forcing  Table , as in mnts Fig. . Therefonts Fig. . In turnAlthough this observational analysis demonstrates the role of the SIOD in the subsequent year’s CP-type El Niño event, it has limitations due to the relatively short period of analysis and the combination of various climate variabilities. Therefore, the impact of the SIOD is further substantiated using idealized experiments with an atmosphere-ocean coupled general circulation model (CGCM) .The SIOD-related SST anomalies persist for longer (roughly five seasons longer) during 1998–2019 than during 1976–1997 Fig. . Consequ00s Fig. , we cond38 43. The monthly mean 925hPa winds and streamfunction were obtained from the National Centers for Environmental Prediction-National Center for Atmospheric Research (NCEP–NCAR) reanalysis 144. The monthly OHC for the upper 300 m was derived from the European Centre for Medium-Range Weather Forecasts Ocean Reanalysis System 5 (ORAS5)45. The analysis period spans from 1976 to 2019 because the period, amplitude, spatial structure, and temporal evolution of El Niño and its relationship with the Indian Ocean SST notably changed after the mid-1970s46.The monthly mean SST data from the Extended Reconstructed Sea Surface Temperature version 5 (ERSST v5)t-tests were used to determine statistical significance47 for regression and correlation analysis. Furthermore, statistical significance testing for the composite analysis was conducted according to the bootstrap method48.Hindcasts of the three-month averages of the Niño indices in Fig. 49 was used for the idealized CGCM experiments. The model resolution was a 1.9° × 2.5° grid with the standard 30 vertical levels for the atmosphere and an approximate 1° grid for the ocean.To investigate the impact of the SIOD on the subsequent year’s ENSO during the winter, the Community Earth System Model, version 1 (CESM1)SST, and A represent the SST in the CGCM, and the conventional dynamical and physical terms to lead SST tendency, respectively. The second term on the right-hand side is the nudging term. By adding the nudging term, the simulated SST always moves toward the observed SST. The nudging time scale (τ) was prescribed as 1 day so that the simulated SST state in the southern IO was strongly nudged toward the given observed SST state.We first performed two partial nudging experiments. The nudging scheme is one of the simplest data assimilation techniques and can be described as follows:In the first experiment, the positive SIOD-related SST anomalies were nudged in the southern IO  with the seasonally varying climatological SST obtained from ERSST v5 for 1998−2019, whereas the other oceans were run freely (Exp_C_SIOD). The second experiment was used as a control, which was identical to the former experiment except that the observed climatological SST anomalies were nudged in the southern IO (Exp_C_CTRL). To obtain the positive SIOD-related SST anomaly pattern, we first collected the years in which the OND SIOD index was larger or small than one standard deviation for the composite analysis. For these years, we calculated the difference in the composite between the positive and negative SIOD events, and then the values were divided by 2 to obtain the final SIOD-related SST anomaly pattern. Finally, the composited SST anomalies were added to the climatological SST. A total of 30 ensemble members with different initial conditions were employed for both experiments. Each experiment was run for 17 months from October (0) to February (+2).37. We first collected the years for which the NCT or NWP index during the winter was greater (less) than 0.5 °C (−0.5 °C). Note that the NCT and NWP indices were calculated using a mathematic rotation of Niño3 and Niño4 indices. Of these years, an EP El Niño (La Niña) year was defined as when the NCT index was greater (less) than the NWP index. We then calculated the difference in the composite between the EP El Niño and La Niña cases and the values were divided by 2 to obtain a final EP ENSO pattern. The CP ENSO pattern was also obtained using the same method for the EP ENSO except that a CP El Niño (La Niña) year was defined as when the NWP index was greater (less) than the NCT index. A total of 20 ensemble members with different initial conditions were employed for both experiments. Each experiment was run for five months from October (0) to February (+1).Second, we performed two additional partial nudging experiments to investigate the impact of the westward-shifted ENSO on SIOD variability after the 2000s. These were identical to the SIOD experiments except that the EP and CP El Niño-related SST anomalies were nudged in the tropical Pacific  . We followed the definitions of the EP and CP ENSOs from a previous study50 was used for the idealized AGCM experiments to investigate the impact of the SIOD on the wind anomalies over the equatorial western Pacific. The model had a horizonal resolution of 1.9° × 2.5° with 30 vertical levels. Two AGCM experiments were conducted: one was a control (Exp_A_CTRL) and the other was a sensitivity experiment (Exp_A_SIOD). Exp_A_CTRL was forced by the monthly climatological SST (1976–2019) over the globe. Exp_A_SIOD was identical to Exp_A_CTRL except that the positive SIOD-related SST anomalies were prescribed in the southern IO . To obtain the positive SIOD-related SST anomaly pattern, the observed SST anomalies were linearly regressed on the OND SIOD index from 1998–2019; subsequently, these regressed SST anomalies were added to the climatological SST field eld Fig. . A totalSupplementary InformationPeer Review File


This study aims to investigate the clinical application and feasibility of C2 subfacetal screws in patients with Klippel-Feil syndrome (KFS), narrow C2 pedicles, and high-riding vertebral arteries (HRVAs).The clinical data of seven patients with KFS, atlantoaxial dislocation, narrow C2 pedicles, and HRVAs treated with C2 subfacetal screws were analyzed in this retrospective study. The internal height, isthmus height, and pedicle width of C2 vertebra were measured using preoperative computed tomography (CT). Subfacetal screws were inserted for 7 patients (12 sides). The position and length of the screws were observed using postoperative CT. Intraoperative dura mater and vertebral artery (VA) injuries were recorded. Bone fusion was observed using follow-up CT.The internal height was 10.5 ± 3.2 mm, the isthmus height was 3.7 ± 1.8 mm, the pedicle width was 3.0 ± 1.4 mm, and the screw length was 19.7 ± 1.5 mm. All patients had HRVAs and narrow pedicles. No injury to the dura mater and vertebral artery (VA) occurred in this group of patients. Bone fusion was achieved in all patients during follow-up.In patients with KFS, HRVA, and a narrow C2 pedicle, there is sufficient space below the C2 articular surface for screw insertion. When the pedicle is narrow and the C2 pedicle screw is not suitable for placement due to possible injury to the VA, subfacetal screws are a feasible alternative. Klippel-Feil syndrome (KFS) refers to congenital cervical segmentation failure, which is a common bone structure deformity in the craniovertebral junction . Common The pedicle screw is the most effective and commonly used fixation technique for the C2 vertebra . A high-Existing C2 screw alternatives include improved pedicle screws, lamina screws, and transarticular screws. However, the morphology of a C2-3 fusion is quite different from that of a normal C2 vertebra, and the aforementioned techniques cannot be used to fix the axis safely and effectively in some cases. In 2014, Patkar et al. reported the use of C2 subfacetal screws , which aThis retrospective study analyzed the data of seven patients with KFS, with associated AAD, narrow pedicles, and HRVA, who underwent subfacetal screw insertion. Our study assessed the feasibility and effectiveness of C2 subfacetal screw insertion for these patients. The details of the clinical application and surgical technique of subfacetal screw insertion are reported.From June 2017 to April 2021, seven patients with C2-3 fusion with associated AAD, narrow pedicles, and HRVA presented to our department. These patients received C2 subfacetal screws insertion (12 sides), and all procedures were performed by the senior author of this study (F.T.). The clinical features of these patients are summarized in Table Chamberlain line invasion and the atlantodens interval (ADI) were measured using midsagittal computed tomography (CT). Congenital AAD was diagnosed when the ADI was more than 3 mm; basilar invagination (BI) was diagnosed when chamberlain line invasion was more than 5 mm .During preoperative parasagittal CT , we measured the internal height and the isthmus height of the C2 vertebra. The pedicle width was measured using axial CT. Narrow pedicle was considered when the maximum width of the pedicle did not exceed 4.0 mm . HRVA waAll patients in this study were treated with C1-2 joint distraction and occipitocervical fixation. They were placed in a prone position under general anesthesia, with the head fixed in the neutral position using the Mayfield headrest. The skin and subcutaneous layers were incised along the posterior median, exposing the squamous part of occipital bone superiorly and the C2 spinous process and vertebral lamina inferiorly. Thereafter, the C2 nerve root and venous plexus were lifted cranially under the microscope with a nerve stripper to reveal the lateral mass joint. Using the interfacet release and direct distraction technique described in previous research , the artIn patients with narrow pedicles, we chose to insert a C2 subfacetal screw. The chosen entry point was approximately 3 mm to 4 mm below the midpoint of the posterior border of the C2 superior articular surface, with the entry point parallel to the superior articular surface or slightly downward, with a medial inclination of approximately 20 degrees. Screws were inserted into the C2 vertebral body as far as possible. The chosen screw length was 22 mm or 24 mm and the depth of screw insertion was based on CT image measurements obtained before surgery  (Fig. Patients in this study had an average internal height of 10.5 ± 3.2 mm and an average isthmus height of 3.7 ± 1.8 mm. All seven patients had a HRVA, caused by a reduction in the isthmus height without internal height decrease. The average pedicle width was 3.0 ± 1.4 mm, with 12 out of 14 sides having widths less than 4 mm (Table In all the patients included in this study, C2 subfacetal screws were placed on 12 sides. The length of the subfacetal screws was 19.7 ± 1.5 mm. There was no spinal cord injury or VA injury during the operation (Table C2-3 congenital fusion is often associated with AAD, which results in neurological dysfunction and requires reduction and fixation to relieve ventral cervical cord compression , 15. PedPreoperative measurements of the patients in this study showed a decrease in the isthmus height and pedicle width of up to 3.7 ± 1.8 mm and 3.0 ± 1.4 mm, respectively. According to the diagnostic criteria for HRVA proposed by Neo et al., namely,Presently, commonly used alternatives to pedicle screws and transarticular screws include lamina screws, which are inserted through the junction of the spinous process and lamina. This technology is widely used, but when the height of the spinous process and the thickness of the axial lamina are small, its use is limited. Moreover, this technology only fixes the posterior single column structure and has a relatively weak biomechanical performance and a low fusion rate . RusconiOn the contrary, Patkar et al. and Salunke have used subfacetal screws for C2 screw placement , 20. We All patients in this study had AAD. Intrafacet distraction and implantation of the cage were performed during the procedure. The following details were given utmost attention during the procedure. First, the screw path of the C2 subfacetal screw is located on the upper and inner sides of the VA. If the screw loosens and subsidence occurs after surgery, VA injury would occur. Thus, a cage for inter-articular fusion was inserted to reduce the stress of screw and prevent subsidence. Second, because of the entry point of the C2 subfacetal screw close to the articular surface and the caudal tilted trajectory, the cage should be inserted first, and thereafter, the screw should be inserted. If the screw is inserted first, the path of the cage entering the lateral joint may be blocked by the screw head, which is not conducive for the preservation of the C2 nerve root. We observed a large operating space around the lateral articular space after cage implantation; therefore, there was no need to cut the C2 nerve root during the procedure. Third, examination images should be carefully studied before the procedure, with special focus on the internal height measurement and design of the screw path. The direction of the screw path should not be too downward to prevent the screw from entering the transverse foramen. The tapered tap should be carefully inserted during the puncture of the screw path. After inserting the tapered tap at 1 mm to 2 mm, the operator should retreat to observe the bleeding. After entering the tapered tap at a depth of 10 mm, it can be determined to exceed the vertebral artery, and then it is safe to advance the tapered tap. In one patient, a screw invaded the transverse foramen during the procedure, but no obvious bleeding was observed when the awl was used for puncture during the procedure; however, when the screw was inserted, it burst the screw path and partially entered the transverse foramen. We adjusted the screw placement direction inward and upward, and the vertebral artery was not damaged after screw placement.The reduction in AAD and improvement of clinical symptoms were achieved for all patients in this study Figs. , 5. We cHowever, this study had some limitations. There were only seven patients included in the study. Studies involving more patients or a larger number of participants are needed to confirm the efficacy and safety of this procedure. In addition, during this study, the average internal height of the C2 vertebra of the seven patients was more than 4 mm and the VA was far from the superior articular surface; therefore, there was enough space for subfacetal screw insertion. However, if the internal height was small and the VA was closer to the superior articular surface of C2, then this technique would not be appropriate. Therefore, more studies are needed to confirm whether HRVA are caused by isthmus height loss in patients with KFS. Additionally, the follow-up time was short, not allowing for observation of the long-term effects of inserted subfacetal screws. Therefore, studies with longer follow-up duration are needed to sufficiently monitor possible long-term effects. The trajectory length of the subfacetal screws was smaller than that of pedicle screws; therefore, the biomechanical properties may be inferior to that of pedicle screws.When C2-3 fusion is associated with HRVA or pedicle stenosis, there is sufficient space below the C2 articular surface for screw placement. The C2 subfacetal screw is a viable alternative treatment for KFS with pedicle stenosis.


Staphylococcus aureus allows for the continuous production of cell walls even after the inactivation of intrinsic PBPs. Thus, the discovery of novel therapeutics with enhanced modulatory activity on PBP2a is crucial, and plant secondary metabolites, such as phenolics, have found relevance in this regard. In this study, using computational techniques, phenolics were screened against the active site of PBP2a, and the ability of the lead phenolics to modulate PBP2a’s active and allosteric sites was studied. The top-five phenolics (leads) identified through structure–activity-based screening, pharmacokinetics and synthetic feasibility evaluations were subjected to molecular dynamics simulations. Except for propan-2-one at the active site, the leads had a higher binding free energy at both the active and allosteric sites of PBP2a than amoxicillin. The leads, while promoting the thermodynamic stability of PBP2a, showed a more promising affinity at the allosteric site than the active site, with silicristin  and epicatechin gallate  having the best affinity at the active and allosteric sites, respectively. Interestingly, the modulation of Tyr446, the active site gatekeeper residue in PBP2a, was noted to correlate with the affinity of the leads at the allosteric site. Overall, these observations point to the leads’ ability to inhibit PBP2a, either directly or through allosteric modulation with conventional drugs. Further confirmatory in vitro studies on the leads are underway.The acquisition of penicillin-binding protein (PBP) 2a in resistant strains of  Staphylococcus aureus is a Gram-positive bacterium that has remained a major public health concern over the years, due to its resistance to major antibacterial therapies [S. aureus (MRSA) is currently one of the most common global causes of infective endocarditis, as well as skin and respiratory system infections [herapies . For insfections ,3. In mafections ,4. Howevfections ,4,5,6,7.S. aureus, it is a five-chain peptide L-Ala-γ-D-Glu-L-Lys(Gly)5-D-Ala-D-Ala with the lysine side chain being linked to pentaglycyl during cell wall crosslinking [The survival of bacteria is dependent on the integrity of their cell walls, which are made up of peptidoglycan as the primary building unit ,9. Peptislinking . The traslinking ,10.Staphylococcus aureus possesses four inherent PBPs  [mecA gene, a fifth PBP known as PBP2a is frequently found in MRSA, conferring antibiotic resistance in the organism [S. aureus is a high-molecular-weight class B PBP identified solely in antibiotic-resistant strains of S. aureus. Penicillin-binding protein 2a’s transpeptidase active site, like that of other PBPs, contains the active site serine (S403) at the N-terminus of the SXXK sequence motif [nd PBP4) . Howeverorganism . As a reorganism ,13. Penice motif ,14,15. Ice motif ,16. As ace motif ,16. Alloce motif . Howeverce motif ,13,14,16S. aureus) [S. aureus. Thus, with over 10,000 phenolics isolated over the years and reported in the literature with different pharmacological characteristics [S. aureus. The most promising phenolics were further studied for their thermodynamic compatibility with PBP2a and their ability to modulate the active site of PBP2a following binding at the allosteric site  . These c aureus) ,17. Phen aureus) ,17, whil aureus) . In addi aureus) ,20,21. Teristics , we emplric site . This waS. aureus (3ZFZ) was obtained from the protein data bank (PDB) . The structure was prepared via removal of water molecules and nonstandard amino acids in preparation for molecular docking using the UCSF Chimera v1.15 software tool [The X-ray crystal structure of PBP2a of are tool . The x-yare tool  and afteare tool .http://zincpharmer.csb.pitt.edu, accessed on 21 August 2021). For the screening, the 3D structure of PBP2a from S. aureus and a consensus phenolic pharmacophore (derived from 32 phenolics) produced from PharmaGist  were used [S. aureus  v 0.9.5. . Before S. aureus (3ZFZ). The superimposition showed that the top-five phenolics and amoxicillin achieved the same orientation with the native inhibitor of 3ZFZ with a low RMSD value of <1, which validated the docking scores observed in the study; these are presented in However, as molecular docking methods often produce pseudo-positive binding conformations as the most-energy-minimised pose, validation of docking studies is often required. One of the most common ways to evaluate the correctness of docking geometry is to measure the root-mean-square deviation (RMSD) of the ligand from its reference position in the answer complex after optimal superimposition . A low Rhttp://swissadme.ch/index.php, accessed on 15 October 2021) and Molinspiration  toolkits were used in this study for a robust prediction of hit phenolics’ physicochemical properties, pharmacokinetics, drug-likeness, and medicinal chemistry friendliness, while the toxicological profiles were evaluated using the Protox II webserver . Taken together with the binding affinity of the top-twenty phenolic compounds, the results of these investigations were utilised to narrow down the list to five compounds.The SwissADME web  was used to molecularly fingerprint the top-twenty compounds. In a nutshell, Galaxy Europe’s “molecule to fingerprint” tool was used to convert the compounds’ smile format into “Open Babel FP2 fingerprints.” The “Open Babel FP2 fingerprints” were then clustered using the fingerprinting algorithms “Taylor-Butina” and “NxN clustering”, with thresholds of 0.8 and 0.0, respectively [Galaxy Europe (The MD simulation was performed as previously described . To summbind = Gcomplex − (GReceptor + Gligand). The PBP2a-ligand plots, which indicate molecular interactions between ligands and amino acid residues at the PBP2a active site or allosteric site, were identified from the final complexes obtained and visualised with Discovery Studio v 21.1.0  [The post-dynamic analysis was performed by saving the systems’ coordinates and trajectories throughout the simulation period and then evaluating them post-simulation using the PTRAJ module of the AMBER 18 package. Exploiting the CPPTRAJ module of the same package, the analysis of root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), the radius of gyration (ROG), and solvent-accessible surface area (SASA) were carried out and their plots were generated using Origin v 6.0 . LikewisCA, USA) .S. aureus , ZINC71621503 (propan-2-one), ZINC38337968 , ZINC95486076 (Chroman-4-one), and ZINC03978503 ) passed the Lipinski rule of five . LipinskS. aureus than amoxicillin. Silicristin, epigallocatechin 4-benzylthioether, chroman-4-one, and epicatechin gallate, when complexed with PBP2a at the active site had −25.61 kcal/mol, −24.75 kcal/mol, −22.34 kcal/mol, and −23.11 kcal/mol, respectively, which were all higher than the −21.54 kcal/mol observed with amoxicillin, with silicristin having the highest binding free energy score (The binding free energy measures the difference in energy between a complex (ligand and receptor) and its unbound receptor component and the higher the negative value, the better the ligand’s affinity for the protein . In thisgy score . This obS. aureus was studied by evaluating the RMSD, ROG, SASA and intramolecular numbers of hydrogen bonds and distances over a 120 ns simulation time. The RMSD trajectory depicts the time-dependent departure of a complex’s structure from its unbound structure and the lower the RMSD value, the closer the complex structure to the unbound structure which depicts stability [S. aureus PBP2a when bound at its active site relative to the reference β-lactams investigated. Notably, silicristin having a higher RMSD value than amoxicillin when complexed with PBP2a, suggestive of silicristin’s lesser thermodynamic stability with PBP2a did not support the higher binding free energy observed for silicristin relative to amoxicillin in this study, however, this observation could be attributed to the sudden drop in stability (the increase in RMSD) after 60ns as observed from the RMSD plot  inside the active site and realigns the conformation of key residues to allow catalysis . DockingS. aureus than conventional β-lactam antibiotics . Interestingly, epicatechin gallate and other top-five phenolics which demonstrated their potential affinity for the allosteric site of PBP2a in the order of epigallocatechin 4-benzylthioether > silicristin > chroman-4-one > propan-2-one, all had a higher binding free energy at the allosteric site than at the active site. This observation demonstrates the better potential affinity of the top-five phenolics and most especially epicatechin gallate for the allosteric site, revealing their potential benefit in treating S. aureus infections in combination with standard β-lactam antibiotics.The binding of the top-five phenolics , propan-2-one , epigallocatechin 4-benzylthioether , chroman-4-one , and epicatechin gallate ) with PBP2a resulted in a higher binding free energy than the amoxicillin  complex at the allosteric site. This observation, while corroborating the molecular docking scores at the allosteric site, further demonstrates the potential affinity of the top-five phenolics for the allosteric site, revealing their probable capability to modulate the amino acid residues of PBP2a allosteric site. Interestingly, this finding is in correlation with the observation of Alhadrami et al.  where sobind: −47.65 kcal/mol; RMSF: 3.61 Å) > epigallocatechin 4-benzylthioether , silicristin  > propan-2-one  > Chroman-4-one  > amoxicillin ) and the graft is presented in The allosteric modulation of the active site gatekeeper residue of PBP2a was studied by evaluating the RMSF of PBP2a through a 120 ns simulation time. Except for epigallocatechin 4-benzylthioether, binding at the allosteric site of PBP2a by the top-five phenolics (silicristin (2.45 Å), propan-2-one (2.49 Å), chroman-4-one (2.55 Å), and epicatechin gallate (2.06 Å)) and amoxicillin (2.56 Å) resulted in a lesser average RMSF than the unbound PBP2a (2.71 Å), with epicatechin gallate having the lowest RMSF value . This obS. aureus  relative to epicatechin gallate despite the high number of interactions. Moreover, epicatechin gallate at the allosteric site of PBP2a (11 interactions), interacted with a relatively shorter bond length (3.87 Å) when compared with epigallocatechin 4-benzylthioether and other studied compounds , Van der Waals, amide π-stacked, π-anion, π-sigma, π-cation, π-π t-cation, π-π t-shaped, π-π t-stacked, π-alkyl, alky as well as some unfavourable donor–donor interactions . The PBPompounds . In addiompounds . All theompounds . PBP2a +ompounds a at the S. aureus. Specifically, the lead phenolics showed more promising affinity at the allosteric site than the active site, with silicristin and epicatechin gallate having the best affinity at the active and allosteric sites of PBP2a, respectively. Interestingly, the modulation of Tyr446, a residue implicated in impeding access of β-lactams to the PBP2a active site, was noted to correlate with the affinity of the lead phenolics with the allosteric site of PBP2a with epicatechin gallate, which has the highest binding free energy, resulting in the highest fluctuation of Tyr446. This observation is indicative of the lesser involvement of Tyr446 in the intramolecular interactions of adjacent residues of PBP2a, thereby creating lesser obstructions of the PBP2a active site. Furthermore, when the lead phenolics bind at the active site of PBP2a, Tyr446 had less fluctuation, implying its active involvement in the binding of the compounds. These observations are suggestive of the possible advantage of the lead phenolics in the direct and synergistic treatment of infections caused by S. aureus and, interestingly, as the lead phenolics had favourable pharmacokinetics and a feasible synthetic score, signifying the possibility of structural alterations for improved druggability and toxicity. However, further in vitro and in vivo confirmation of the activities observed in this study is strongly recommended and efforts are underway in this direction.Findings from this study have identified lead phenolics with the capability to bind to the active and allosteric sites of PBP2a of


The development of strong, well-conformed hooves begins pre-natally and is on-going throughout the horse’s life. This paper explores specific aspects of the development and adaptation of the distal forelimb in Thoroughbred foals with emphasis on how the hoof responds to weight bearing early in life. The thickness of the epidermal tissues at the toe increased from 2.84 ± 0.41 mm before birth to 4.04 ± 1.10 mm by 4 months of age. The increase in thickness was accompanied by decreased density of horn tubules at the toe while the number and size of horn tubules increased at the medial and lateral quarters. This provides a malleable hoof capsule with increased resistance to fracture toughness at the quarters to allow for skeletal growth. Between 4–6 months of age, the hoof widens, and higher loading on the medial side (>60%) vs. the lateral side (<40%) may be factors that influence mature asymmetric hoof shape. Shortly after 12 months-of-age, the dorsal hoof wall angle becomes parallel with the dorsal wall of the distal phalanx, thus optimizing the functional capacity of the hoof capsule in the weanling Thoroughbred.Conformation of the hooves and distal limbs of foals and factors influencing their morphological development have not been reported in detail for the Thoroughbred breed. In this paper we explore morphogenesis of the equine distal limb in Thoroughbred foals with emphasis on adaptations in response to weight bearing early in life that prepare the foal for an athletic career. Novel data from four studies are presented chronologically during key time periods to illustrate specific aspects of distal limb growth and adaptation. Dorsal epidermal thickness increased from 2.84 ± 0.41 mm in utero to 4.04 ± 1.10 mm by 4 months of age. The increase in thickness was accompanied by decreased tubular density, increased inter-tubular material, and an increase in number and size of tubules at the quarters, which provided a malleable hoof capsule to allow for skeletal growth. Between 4–6 months of age, the hoof widens, and higher loading on the medial side (>60%) vs. the lateral side (<40%) may be factors that influence mature asymmetric hoof shape. Shortly after 12 months-of-age, the dorsal hoof wall angle and dorsal parietal angle of the distal phalanx become parallel, thus optimizing the functional capacity of the hoof capsule in the weanling Thoroughbred.  Horses belong to the taxonomic order Perissodactyla which is distinguished by having an odd number of toes; in horses a single weight-bearing toe is derived from the third digit. Furthermore, horses are unguligrade implying that the metapodials and digits are elevated from the ground. As a result, weight-bearing has become the responsibility of the tip of the third digit encased by the modified nail or ungula. The hoof consists of an outer protective layer called the hoof capsule derived from the nail and composed of insensitive cornified tissues. Inside the hoof capsule are sensitive tissues including bone, ligament and tendon that are part of the locomotor apparatus.The horse’s outer appearance or conformation is aesthetically and functionally important with particular aspects of conformation being prioritized in specific breeds. Correct digital alignment and conformation are emphasized in Thoroughbred racehorses because the high speeds at which they train and race are associated with high impact accelerations ,2 and exIn the sagittal plane, the hoof angle, measured mid-dorsally at the toe should equate to the pastern axis. The resulting hoof-pastern axis is regarded as a crucial conformational feature  with regThe role of the hoof in the hoof-surface interaction of the galloping horse includes assisting in damping impact accelerations and high magnitude ground reaction forces. This is achieved by converting impact energy into strain energy, supporting the limb during maximal loading and providing grip . The strThe hoof wall consists of 3 layers: the stratum externum consisting of the thin layer of periople, the stratum medium characterized by its tubular and inter-tubular horn structure, and the stratum internum with its inwardly projecting lamellae that interdigitate with corresponding dermal lamellae from the distal phalanx . The strThe strong rigid hooves that are necessary for supporting body weight during locomotion pose a risk of damage to the intra-uterine tissues during gestation and to the birth canal during parturition. To reduce the risk of injuring the mare, the fetus develops soft, protective ‘slippers’ that form a gelatinous deciduous hoof capsule  that proConformational differences between foals and mature horses are evident in the external features of the hoof and underlying skeletal structures. However, limited information has been reported to date on factors that influence hoof growth and development either in Thoroughbreds or other breeds. Four studies were carried out by Curtis  to documFour independent studies were conducted to evidence changes in hoof shape in this population using convenient sampling of Thoroughbred fetuses and foals ranging from 38 days pre-partum to 503 days post-partum.The subjects were 15 Thoroughbred cadaver fetuses and foals, aged −38 to 134 days, that had died naturally or were still born, none had been euthanized. All subjects had healthy limbs and hooves. After disarticulating the digits at the metacarpophalangeal joint, they were frozen within 2 h after death.The hooves were sectioned parallel to the coronary band, midway between the coronary band and the bearing border. Tissue samples were collected from the hoof wall on the dorsal, lateral and medial sides, as described by Reilly et al. . The sam2).Two parallel lines, 1 mm apart, were added to each image using Photoshop 7.03 . A single image showing the full width of the hoof wall was created by concatenating sequential images . EpidermTwenty-two Thoroughbred foals that were born during the same year (2011) at three stud farms located in close proximity and using the same farrier and veterinary practice. None of the foals had obvious conformational faults or had been treated for lameness and all foals were handled daily by experienced handlers. During data collection, the foals and their dams were controlled by experienced handlers. They were habituated to and monitored throughout the measurement procedures to ensure that no undue stress was caused. An experienced radiographer collected and processed the radiographic images using standard procedures. All foals were measured on the same day in the months of June when they were 33 to 145 days old and November when they were 180 to 296 days old. The reasons for selecting data from these months were that in June the foals were at least a month old and after November some foals would be sold and not available to include in the study. All of the foals’ had their hooves trimmed by the same farrier according to a published protocol  and noneA line was drawn in indelible ink through the middle of the frog to the point of toe to establish the midpoint of the dorsal wall . The solThe foals’ fore hooves were then placed upon two wooden blocks, 50 mm high, on a level floor with the metacarpal segments vertically aligned . StandarTwenty-eight Thoroughbred foals born in 2013 (age 13 to 77 days at the start of the study) were selected from two stud farms on the basis of being healthy and having no behavioral difficulties. For these foals, data were collected when acquired flexural deformities (AFDs) are reported to emerge. Foals included in this study were categorized visually according to the presence or absence of AFD. Foals in the AFD group were treated by trimming the heels, complete stable rest for up to 2 weeks and, in two cases, a toe extension was applied. An additional twelve Thoroughbred weanlings born in 2013 (285 ± 26 days at the start of the study) were selected to extend the hoof growth and compression dataset with the following criteria; healthy foals born at two stud farms during the same year, that had no history of lameness. The management involved daytime turnout on pasture with overnight stabling and supplementary feeding of a well-balanced diet. They were handled by experienced personnel at all times, including during data collections. Immediately prior to each measurement, the same farrier trimmed the feet and rounded the distal border to a radius of approximately 3 mm without rasping the outer hoof wall.The coronary hairline was defined with red indelible ink. The mid-dorsal centre of the hoof wall at the bearing surface was located as described in With the hoof lifted and extended, five photographs were taken using a digital camera with the lens perpendicular to the dorsal hoof wall from a distance of 0.3 m. Lateral and medial photographs were taken in a similar manner. The photographs were observed immediately to check they were correctly framed and focused. The best image was selected and downloaded to a software package  designed to extract hoof measurements . Repeat Linear dimensions of the hoof were measured three times each using the circular disc for calibration and the data were recorded in Excel. Toe length was measured between the coronary hairline and bearing border. The distance from the coronary hairline to the proximal acrylic reference point was measured to determine hoof growth and hoof growth was divided by the number of days since the previous evaluation to calculate hoof growth rate. The distance between the two acrylic points at the first data collection was used as a reference for measuring hoof compression. Compression rate was calculated as the amount of compression divided by the number of days between measurements. From these measurements toe length, hoof growth rate and hoof compression rate were determined at each data collection.The 28 Thoroughbred foals selected for growth and compression measurements were also used to measure solear load distribution. After the fore hoof photographs had been taken, they were walked onto a pressure mat which recorded the loading pattern at 50 Hz for 20 s during standing. No attempt was made to move or alter the foals’ stance after they stopped on the pressure mat. Three recordings were made and saved in Footscan Balance. The recording that showed the smallest amount of sway was chosen for analysis and frame 500 from that recording was used for analysis. An image from this frame was extracted from Footscan Balance and the dorsopalmar axis of each hoof was determined in the horizontal plane using Photoshop 7.03 using the pressure contours of the frog and external hoof features to align the axis.The selected trial was exported to Excel and the chosen frame was used to obtain an image of each loaded cell from which the left and right force values were calculated. Cross hairs positioned at the geometric centre of the data were overlaid on the force data and rotated to align with the hoof’s dorsopalmar axis .The force values in each  were calculated by summation of the forces for all loaded cells in the quadrant . Where tData from all four studies were combined into one spreadsheet in SPSS 28.0.1.1 . Study, foal identification, age, limb and measured variables for each study were input. The data were then separated by age, based on the following criteria: −38 days to birth (non-weightbearing), 1 day to 4 months of age (based on the emergence of AFD ), 4 to 6p < 0.05. Where significant differences were found, Bonferroni post hoc pair-wise comparisons were performed to identify which regions differed significantly.For epidermal features, regional differences in structure were compared using a General Linear Model of covariance with foal as a covariate and significance set at p < 0.05).For skeletal conformational features, descriptive statistics (mean ± standard deviation) were calculated for each available period. In addition, to illustrate the sagittal plane conformational changes over time, the slope of the DHWA and dorsal parietal angle of the distal phalanx, and the slope of the proximal integument depth and distal integument depth were compared for statistical significance   , though  < 0.05) , integumIn contrast to the normal development described above, some foals develop AFD in which the digit becomes angled more steeply and, in severe cases, the heels may even be raised from the ground , which sn = 18) and AFD (n = 8) conformation. Load distribution was indeed significantly higher at the toe in foals showing early signs of AFD (p < 0.05)  . Hoof grThoroughbreds are reported to completely renew the fetal hoof capsule between 120 and 165 days after birth  and manyp = 0.148, η2 = 0.137). In the control group, loading was significantly different (p < 0.001) between all three regions, and highly significantly different comparing medially to laterally. Widening of the solear surface together with continued evidence of greater loading medially vs. laterally  = 2.061, aterally  may be iaterally . PlasticBy 6–12 months of age, hoof length in the Thoroughbred foal is typically 95 ± 9 mm, hoof growth rate at the dorsal wall is 0.24 ± 0.08 mm/day, hoof compression rate is −0.01 ± 0.01 mm/day and the DHWA is moving closer towards the typical angles found in mature horses. During this time the shape of the hoof capsule changes from resembling an inverted cone in the foal to an obliquely truncated cone in the mature horse ,32. Thisp < 0.001) the two surfaces became more parallel over time. The slope of the regression lines for the proximal and distal integument depths were also significantly different leading to a relatively greater increase distally and a more parallel alignment between the dorsal hoof wall and the dorsal surface of the distal phalanx. The angles were calculated to converge at 391 days when the DHWA is 49.5 deg.The relative reductions in DHWA and dorsal parietal angle and in the proximal and distal integument depths are illustrated in Post-partum, the hoof epidermis is generated by and grows distally from the coronary band , which rThe DHWA should also be parallel to the dorsal parietal angle of the third phalanx in mature horses and changes in the distal integument depth may be indicative of serious health concerns, such as laminar separation . With thThis manuscript defined structural and conformational changes in the Thoroughbred digit and hoof from late gestation to one year of age and loading patterns that may influence these changes. The data were analyzed in a chronological manner to illustrate how weightbearing and growth influence shape changes over this period.In foetal hooves, the epidermis is thicker at the dorsum than at the quarters due to the presence of a larger number of horn tubules with a larger diameter, providing a structure with features of a mature horse to prepare for weightbearing.In young foals, epidermal thickness at the dorsum increases, with the greater thickness being ascribed to an increase in inter-tubular horn rather than an increase in tubule number or size. Pressure within the interpapilliary space stimulates even hoof growth , which mAt this age, some foals developed AFD with a ‘heels up’ posture that suggests a dorsal shift in loading. This study confirmed that foals with AFD have greater loading at the toe than foals with normal limb posture. As AFD can lead to club foot, the non-significant differences in hoof growth, which were due to an under powered sample, warrant further investigation. Based on these initial findings, early identification and treatment of AFD is essential for a favorable outcome.Between 4 and 6 months of age, the foetal hoof wall is completely replaced, the width of the hoof expands and the asymmetric loading patterns in standing that have been recorded since the start of weight-bearing continue to be evident. The shape of the hoof becomes an obliquely truncated cone, which indicates that the internal stresses will now allow the hoof to function in a similar manner to the function of the hoof mechanism in mature horses. These factors may be key in changing the shape of the symmetrical foetal hoof into the asymmetrical mature hoof, but further data is required to confirm this.Throughout the first year of life, the dorsal epidermal thickness increases at a more rapid rate distally than proximally. As a result, the DHWA decreases and becomes better aligned with the dorsal parietal angle of the distal phalanx and the hoof-pastern axis. In Thoroughbreds, parallelism of the hoof wall with the skeleton typically occurs around 391 days, which provides the weanling with an athletic hoof that optimizes functional capacity for galloping.


Icebergs account for approximately half of the freshwater flux from the Greenland Ice Sheet and they can impact marine ecosystems by releasing nutrients and sediments into the ocean as they drift and melt. Parameterizing iceberg fluxes of nutrients and sediments to fjord and ocean waters remains a difficult task due to the complexity of ice-ocean interactions and is complicated by a lack of observations. Acquiring iceberg samples can be difficult and dangerous, as icebergs can break apart and roll without warning. Here we present open source design files for a small, lightweight ice coring drill that can be reproduced using modern computer numerical control (CNC) machining and 3D printing technology. This ice core drill can rapidly acquire small ice samples from icebergs and bergy bits using a standard commercial, off-the-shelf battery-operated hand drill. Design files and a recent field expedition to Northwest Greenland are described. Ice core collection required only 30 s, thereby minimizing risks to scientists. Specifications table:11.1Mass loss from the Greenland Ice Sheet (GrIS) has increased by nearly a factor of six since the 1980s The variability in the biogeochemical composition of icebergs needs to be adequately characterized to develop ecosystem models for polar regions 1.21.2.1Titanic in 1912 Titanic incident spurred interest in iceberg properties to understand their thermodynamic and mechanical properties The International Ice Patrol (IIP) began monitoring iceberg populations in the North Atlantic following the sinking of the In the past few decades, offshore oil exploration in the North Atlantic has renewed interest in iceberg properties from the standpoint of iceberg management More recently, the increased mass loss from the Greenland Ice Sheet has drawn attention to the impacts of icebergs on marine ecosystems. In these studies, small samples (<0.5 m) were obtained either using ice axes or by fishing small bits of floating ice out of the water 1.2.2Ice drilling systems have been used for over a century on glaciers, ice sheets, and in sea ice Commercial products in this size range are also limited, likely due to a lack of demand. The Kovacs Mark II coring system is 1 m long and costs approximately $5000 (USD) Thus, the lack of either a suitable commercial product or an open source design necessitated the development of an ice drill to fulfill our research objectives. Given that a sample of 0.5–1 kg is sufficient for the chemical analyses that motivate this paper, we require a miniature ice drilling system. In addition to collecting samples of a relatively consistent size, a miniature ice drilling system is easier and, perhaps, safer to operate from a pitching, rolling, and heaving small boat.Here we present a small and lightweight ice coring drill design that can rapidly acquire samples from icebergs and bergy bits using a standard commercial, off-the-shelf battery-operated hand drill. The hand drilling system was developed in parallel with a drone-mounted ice sampler We describe the design files and a recent field expedition to Northwest Greenland, where the miniature ice coring drill presented here was used to retrieve 11 samples. Sample acquisition took approximately 30 s, permitting ‘in-and-out’ sampling of freely drifting icebergs using a small boat. This rapid sampling capability reduces risks to researchers and their support equipment and will increase the number of iceberg samples available to develop robust statistical parameterizations of iceberg nutrient and sediment loads.2•Simple to assemble and operate•Fast sample acquisition•Lightweight and smallHere we describe a lightweight and small ice coring drill that is simple to assemble and operate. The pictures of the coring heads in The ice coring drill presented here was created directly from the design files using modern computer numerical control (CNC) machining and 3D printing techniques. We assume, perhaps incorrectly, that those engaged in the acquisition and analysis of ice cores have access to the resources needed to reproduce this ice coring drill, including machinists, CNC tools, 3D printers, and battery-operated hand drills, through their university, research institute, or place of employment.With this assumption in place, the cost of the drill reflects the total cost of the raw materials and does not consider the costs of procuring, maintaining, and operating CNC machines and 3D printers, nor does it include the salary of those responsible for operating the aforementioned equipment as these costs are usually covered by institutional overhead and/or cost-sharing agreements between departments and/or institutions. Should this assumption prove to be incorrect, which is likely in some cases, interested parties can seek out the services of a private machine shop. If a private machine shop is unavailable or too costly, we invite those interested to contact the corresponding author. Furthermore, the authors make no attempt to account for regional differences in the prices of the raw materials. Finally, the cost estimate does not include the cost of a battery-operated hand drill as these drills are versatile pieces of equipment and are included in the toolkit of every expedition.2.1The cutting head , Fig. 2 2.2The main body of the drill is 191 mm long and is composed of inner and outer sleeves. The inner sleeve is a 70 mm diameter aluminium cylinder, which holds the ice sample. The outer sleeve is 3D printed in SLS nylon and uses spiral grooves 2.3The drive shaft consists of a hexagonal bit that is machined from stainless steel and inserts into the chuck of the hand drill. The shaft assembly includes the drill top, which is machined from aluminium, and is threaded to hold the body of the drill. The end stop is machined from aluminum and prevents the ice core from reaching the top of the drill.3The design files include .STL and .STEP files for the 3D printed and machined parts, respectively. There are no metal alloy requirements for the machined parts. The 3D printed parts were constructed using SLS nylon, though they can also be constructed using PLA, PETG, and/or ABS, and the metal parts are machined using aluminium, stainless steel, spring steel, and knife steel, as indicated in the bill of materials table. For the most part, the file names are self explanatory and their placement is indicated in 44.1Before commencing assembly of the drill ensure that the following criteria are met for the manufactured parts. Ensure that the M74  top 13; , drill h top 13; , and dri top 13;  are mach4.2Always refer back to the assembly files provided for the orientation and number of parts. Use caution when handling the core dogs and cutting teeth and always wear protective gloves when handling sharp items.4.3Use a sacrificial M3 self-tapping screw to tap out the screw holes for the cutting teeth 7–9; , core doovers 5; ) into th4.4Ensure that the drill center tube 11;  fits int4.5Ensure that the drill shaft 12;  inserts stop 10; ) onto thstop 10; . Tighten4.6Begin by placing the cutting head assembly on a level surface, with the threaded end facing up. Next, fit the body assembly onto the cutting head assembly by fitting the drill sleeve onto the protruding drill sleeve pins. Gently press down on the drill center tube and rotate it with one hand while holding the drill head with the other to engage the threading. The two should thread together with minimal force. Avoid cross-threading, and repeat this step if in doubt. Ensure that the drill sleeve fits squarely against the drill head with no gaps. Thread the drill center tube into the cutting head assembly until fully seated. With the new assembly stood on a level surface, thread the drive shaft assembly onto the exposed threaded section of the drill center tube. The two should engage smoothly with minimal force. If not, lubricate the threads with a small amount of silicone grease. Thread the drive shaft assembly onto the assembly until fully seated, gripping the drill head with one hand and the drill top with the other. Ensure that there are no gaps between the drill top and drill sleeve and the sleeve is held snugly in place. If not, ensure that the center tube is fully seated in either the drill head or drill top. Lay the drill assembly down on a flat surface, supported enough as to not roll away. Install the cutting teeth by placing them onto the mounting surfaces. Secure each cutting tooth with two M3 55.1•WARNING – icebergs can be unstable and unpredictable. Approach with extreme caution and only under the supervision of experienced personnel.•WARNING – Never hold the drill by its flutes or head when mounted in an electric hand drill; serious injury may follow.•CAUTION – always wear proper protective equipment; protective gloves and safety glasses as a minimum when drilling.•CAUTION – cutting teeth are sharp and should be covered when not in use to avoid injury and/or damage.•CAUTION – Always have a trained second person standing by and attentive when operating the drill.•ATTENTION – Faster drilling speeds have been shown to produce better results. Ensure that the intended electric hand drill can reach minimum 1500 RPM.5.2Drape a plastic bag over the electric hand drill to protect from water damage from ice cuttings. If the ice coring drill is already assembled, proceed to the next section. Align the threads on the ice coring drill top with threads on the body of the ice coring drill. Slowly screw the two parts together and make sure to avoid cross-threading. Screw down snugly. Insert the hexagonal shaft into the bit holder on the electric hand drill. Tighten the hand drill’s chuck to secure the hexagonal shaft. Verify correct rotation - keep clear of people and other equipment. The drill should now be handled with extreme care and treated as dangerous.5.3Place the ice coring drill squarely against the iceberg with all 3 sets of cutting teeth engaged. Begin slowly rotating to form a groove to guide the cutting teeth. After a 5 mm groove has been formed, slowly increase to full speed – faster is better – slow drilling can get stuck . Let the5.4Note that anyone handling the ice sample should wear sterile gloves to prevent contamination if sampling for microbes. Loosen the top of the ice coring drill with a quarter twist. With the electric hand drill set to its slowest speed, slowly unscrew the body of the ice coring drill from the drill top. Make sure to point the cutting teeth away from any people or equipment . Once un66.1The ice coring drill system was tested during the ‘Vaigat Iceberg – Microbial Oil degradation and Archaeological heritage investigation’ (VIMOA). VIMOA took place along the Nuussuaq peninsula (north of Disko Island) from 29 July to 16 August, 2019. VIMOA was a multidisciplinary research cruise that investigated iceberg drift and deterioration, microbial assemblages and microbial oil degradation, and coastal archaeological sites situated near natural oil seeps 6.2While the ice coring drill performed well during sampling, sample extraction was awkward at times. We suggest the following improvements for future versions. First, a device that immobilizes the body of the drill and, therefore the cutting teeth, which do not discriminate between ice and human flesh, while it is unscrewed from the top. Second, a plunger of some sort would be helpful for extracting the ice sample. Finally, a storage sheath should be constructed to protect the cutting teeth during transport. The envisioned improvements could be designed to cover the cutting teeth while immobilizing the drill and still allowing for sample extraction, thereby also making sample extraction safer and easier.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


In the AGEs inhibition assays, quercetin showed significant results in both non-oxidative and (IC50 0.04 mg/mL) and oxidative assays (IC50 0.051 mg/mL). Furthermore, quercitrin showed inhibitory activity in the non-oxidative (IC50 0.05 mg/mL) and oxidative assays (IC50 0.34 mg/mL). A significant inhibition of protein cross link formation was observed by SDS-PAGE analysis. Quercitrin (65%) and quercetin (62%) showed significant inhibition of 15-lipoxygenase. It was thus concluded that flavonoids and other polyphenols present in plant extracts can be effective in management of diabetes and allied co-morbidities.Diabetes mellitus (DM) is a global health concern that is associated with several micro- and macrovascular complications. We evaluated several important medicinal plant constituents, including polyphenols and flavonoids, for α-glucosidase inhibition, AGEs’ inhibitory activities using oxidative and no-oxidative assays, the inhibition of protein cross link formation, 15-lipoxydenase inhibition and molecular docking. The molecular docking studies showed high binding energies of flavonoids for transcriptional regulars 1IK3, 3TOP and 4F5S. In the α-glucosidase inhibition assay, a significant inhibition was noted for quercitrin (IC Diabetes mellitus (DM) is an ailment of the endocrine system that is associated with chronic insulin resistance and progressive exhaustion and death of β-cells in the pancreas that leads to hyperglycemia . At presAdvanced Glycation End products (AGE)s are the products produced via a chain of non-enzymatic reactions between reducing sugars, such as glucose, and the amino functionalities of proteins . This reAn association between inflammation, vascular complications and hyperglycemia has been observed in diabetic persons . The patJuglans regia, Salvadora persica, Syzygium aromaticum, Myristica fragrans, Punica granatum and Azadirachta indica were selected for this investigation.Ayurvedic medicines are mainly based on natural products and may include plants, animals or minerals. They have centuries-old evidence of safety and efficacy . Based oJuglans regia L. (Juglandaceae) is an important medicinal plant that is used for treatment of diabetes mellitus, inflammation and cancer , methanol [50% (v/v)] and acetic acid [10% (v/v)]. Protein cross-linked by AGEs were observed by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) . In thisThe developed gel was stained with Coomassie blue and images were obtained using GelDoc. Finally, the Image J tool was used for the determination of integrated density (IntDen). The integrated density was employed further for the determination of percentage inhibition. The integrated density (ID) was determined as follows:The percentage inhibition of cross-linked AGEs was determined using the formula:The anti-inflammatory activity of test samples was evaluated using a standard protocol . To the The percentage inhibition of enzyme activity was calculated as follows:All experiments were performed in triplicate and results were expressed as mean ± SD. Juglans regia, Punica granatum and Myristica fragrans. Thus, it was concluded that these plant species may be considered as candidates for the management of diabetes mellitus and co-morbidities occurring due to AGEs.Diabetes, a very prevalent metabolic disorder, is an important health problem worldwide. Persistent hyperglycemia results in the development of inflammation and several life threatening complications due to the production of AGEs. Various strategies are being used by researchers to introduce new treatment options that have a dual effect i.e., both blood glucose lowering and anti-AGEs potential. Traditional medicinal plants are considered as effective and reliable alternatives of conventional medical therapy due to proven safety and efficacy. In this investigation, selected medical plants and their major constituents were analyzed using in silico and in vitro models. Interactions of flavonoids and polyphenols were observed with transcription regulators 1IK3, 3TOP and 4F5S. Further in vitro assays presented anti diabetic, antiglycation, and anti-inflammatory activities of constituents including juglone, quercetin, quercitrin, apigenin and 2-phenylethylisothiocyanate from