TP63 (human)/Trp63 (mouse) produces multiple isoforms: TAp63 and ΔNp63, which possess a different N-terminus depending on two different promoters, and p63a, p63b, p63g, p63δ, and p63ε as products of alternative splicing at the C-terminus. TAp63 expression turns on in the nuclei of primordial germ cells in females and is maintained mainly in the oocyte nuclei of immature follicles. It has been established that TAp63 is the genomic guardian in oocytes of the female ovaries and plays a central role in determining the oocyte fate upon oocyte damage. Lately, there is increasing evidence that TP63 mutations are connected with female infertility, including isolated premature ovarian insufficiency (POI) and syndromic POI. Here, we review the biological functions of p63 in females and discuss the consequences of p63 mutations, which result in infertility in human patients.The transcription factor p63, one of the p53 family members, plays an essential role in regulating maternal reproduction and genomic integrity as well as epidermal development.  The p53 family proteins play essential roles in maintaining maternal reproduction and genomic integrity, and these essential roles appear to be conserved across different organisms from invertebrates to mammals. A p63/p73 hybrid gene exists in the genome of unicellular flagellates and functionally acts to preserve the genome stability of germ cells in sea anemones . In addiWhile the separation between p63 and p73 is observed in vertebrates, these proteins retain highly conserved female germline-protecting roles with distinct mechanisms in oocytes . When DNTAp73 was shown to mediate chromosome alignment during oocyte maturation in the ovaries, control ovulation, and manage embryonic development during preimplantation . Aging-rThe ovarian reserve is the pool of primordial follicles, which determines the reproductive lifespan of females. A diminished ovarian reserve (DOR) in women indicates a loss of their reproductive potential and is one of the leading causes of infertility . TAp63 iThe transcriptional p53 family members consist of p53, p63, and p73, which have a common structural backbone composed of transactivation (TA), DNA-binding domain (DBD) and oligomerization domain (OD) with differences at the C-terminal region. Unlike p53, which has an unstructured C-terminus, p63 and p73 contain additional domains at the C-terminus. p63 contains a secondary TA domain, sterile alpha motif (SAM) domain, and transactivation inhibitory domain (TID), whereas p73 contains only a SAM domain and TID A. These three proteins are present as inactive forms before adopting an active conformation following multiple phosphorylation events. When p53 and p73 are inactive, they present as tetrameric structures without any phosphorylation. However, p63 forms a tetramer from two dimers following multiple phosphorylations by kinases  Figure B. These The absence of the N-terminal TA domain is, thus, expected to not only generate N-terminally truncated isoforms inert as a transcriptional factor but also to act as antagonists toward their corresponding isoforms. In addition, alternative splicing at the C-terminus during transcription yields several variants ,11. For TAp63β is generated with exon 13 skipped, and TAp63γ lacks exon 11 through exon 14, which encode the SAM domain and TID. TAp63δ skips exons 12 and 13, while TAp63ε is derived from exons 2 through 10 with a 5’ portion of intron 10 transcribed. Structurally, the three main domains of TAp63  remain folded in a quiescent state. Deletion mutagenesis further suggests that the last 70 amino acids of TAp63α constitute a transactivation inhibitory domain (TID) against the N-terminal TA domain, thereby, forming a closed and inactive conformation .The TID contains two distinct inhibitory subdomains with independent regulatory functions . One subTherefore, the generation of multiple variants at the transcriptional level would influence the transcriptional potentials of p53 family proteins and suggests that the relative ratio among isoforms would reflect the overall activity of each p53 family protein at the cellular level when they are co-expressed locally.Our current understanding of the biological roles of TAp63, commonly referred to as the guardian of the female germline, in the genomic integrity of female germ cells has been mostly obtained using mouse models ,15. In mWhen oogonia undergo meiosis between E13.5 and E16.5, they possess multiple double-strand breaks (DSBs) due to recombination between chromosomes. The expression of p63 becomes suppressed in this time frame, supporting the observation that PGCs in germ cell clusters withstand hundreds of programmed DNA DSBs required for meiotic recombination due to the lack of TAp63 expression. However, 4–10 DSBs induced by γ-radiation treatment or other routes of DNA damage are sufficient to induce apoptosis in postnatal immature oocytes due to the expression of TAp63 . The number of TAp63-positive oocytes and the intensity of TAp63 expression begin to increase from E17.5 to postnatal day 5 (PD5) when most of the oocytes have entered meiotic arrest . After bThis distinct expression pattern of TAp63 in female germ cells during embryonic development and after birth raised questions about whether TAp63 expression is essential for the development of female germ cells. Several groups have demonstrated that ovaries from p63 null embryos developed similarly to wild-type ovaries and exhibited a histologically normal phenotype with primordial follicles, growing follicles that appear following recruitment for ovulation, and corpora lutea . This supports that the notion that TAp63 is dispensable for the development of oocytes, follicles, and ovulation although it is expressed during the narrow time window of germ cell development. Interestingly, PGCs in the ovaries of the human fetus at post-last-menses (PLM) 12 weeks did not express p63; however, p63-positive PGCs gradually increase between PLM12 and 16 weeks .All PGCs reach dictyate arrest by postnatal day 5 (PD5) . It has Since predominantly PD5 primary oocytes express only TAp63α and lack γ-H2AX foci, either all meiotic DNA DSBs undergo repair by PD5 dictyate arrest or those that fail to undergo repair undergo TAp63α-mediated death . WhereasWhile irradiated PD5 oocytes demonstrated a prominent TAp63 phosphorylation shift, irradiated PD0.5 oocytes did not. However, IR-induced γ-H2AX levels increased following IR treatment in both PD0.5 and PD5 ovary lysates, indicating that DNA damage was incurred in both PD0.5 and PD5 ovaries . Thus, oTrp63 gene encoding the transactivation domain-specific N-terminus were resistant to equivalent doses of IR treatment that killed virtually all oocytes of primordial follicles in wild-type ovaries [It has been demonstrated that oocytes in the primordial follicles of mice lacking exons 2 and 3 of the  ovaries . SimilarThe association observed between TAp63α phosphorylation and oocyte cell death was the first to suggest that TAp63α was phosphorylated in response to DNA damage . PhosphoSize exclusion chromatography analysis of purified TAp63α demonstrated that it adopts a dimeric, inactive conformation, suggesting that the activation of p63 may be linked to the formation of tetramers. Analysis of the oligomeric state of TAp63α in ovaries of nonirradiated mice revealed a strong signal in the dimer fraction, with no detectable signal in the tetramer fraction. In contrast, ovaries obtained from irradiated mice revealed a significant signal in the tetramer fraction. Accordingly, TAp63α in non-stressed oocytes is maintained in a dimeric and closed conformation, while DNA damage triggers the formation of tetramers . The significantly higher concentration of TAp63α in nonirradiated versus irradiated ovaries further suggested that the formation of tetramers is not suppressed by keeping the intracellular protein concentration low but actively by domain–domain interactions involving the transactivation inhibitory domain (TID). The SAM domain and DBD were described to not be essential for retaining the dimeric state. Later analyses with secondary structure prediction programs predicted the existence of an α-helix (TA1) and two β-strands (TA2A and TA2B) in the TA domain and a β-strand in the TID. TA2B and TID form an anti-parallel β-sheet with a polar and hydrophobic face, proving that a β-sheet covers the tetramerization interface of the tetramerization domain (TD), thus inhibiting the formation of tetramers . In addiTAp63 is the central integrator for DNA damage signaling in oocytes of primordial follicles ,21,22,23Puma and Noxa and the subsequent binding of the potent apoptosis inducers PUMA and NOXA directly or indirectly to BAX and BAK as established by Kerr et al. [TAp63α in its active tetrameric conformation acts as a transcriptional activator, resulting in the induction of r et al. ,28,29. Rr et al. . Additior et al. .Treatment of mouse ovaries with the CHK2 inhibitor II BML-277 to prevent phosphorylation at S582 and subsequent irradiation revealed the nearly complete suppression of TAp63α and almost no formation of tetramers, thus, confirming the previously reported role of Chk2 in the activation process . AdditioPrevious analyses of antibody-purified TAp63 showed clusters of phosphorylation sites in the linker regions between the TA domain and the DBD, between the TID and the SAM domain, and immediately following the CHK2 site, N-terminally to the TID . In addiAll phosphorylation events up to S591 and S592 were deemed essential for efficient tetramerization with the charge density in the region directly N-terminal to the TID as the decisive factor. This stretch of phosphorylated amino acids required for the activation process is located in an unstructured loop directly preceding the TID. Located adjacently is a loop following the TAD harboring three negatively charged aspartate residues that function as the sensor domain for creating the electrostatic repulsive force leading to disruption of the previously established inhibitory β-sheet. Thus, CHK2 acts as a “priming kinase” at S582 and CK1 as an “executioner kinase”, which adds phosphate groups at four consecutive sites  . PhosphoThe tetramerization kinetics of TAp63α following irradiation was revealed to have a sigmoidal time response, where CK1 phosphorylates TAp63α in a sequential manner and a biphasic kinetic. The first two phosphorylation events are fast, while the third one is the slowest and essential for the formation of the open and active conformation. The decrease in phosphorylation kinetics with higher phosphorylation levels may be due to a decrease in the binding affinity of the increasingly phosphorylated peptide, rendering binding in the substrate-bound position as needed for phosphorylation of S591 as less favorable .Infertility, also referred to as subfertility, is defined as “a disease characterized by the failure to establish a clinical pregnancy after 12 months of regular, unprotected sexual intercourse or due to an impairment of a person’s capacity to reproduce either as an individual or with his/her partner” by the World Health Organization (WHO) International Committee for Monitoring Assisted Reproductive Technology (ICMART) and fertility societies ,36. CommFMR1, BMP15, PGRMC1, and ZP3 [The genomic basis of female infertility is rather complex and is determined by many factors. Using karyotype analysis, several chromosome aberrations have been identified to be related to female fertility. Recent studies have found additional candidate monogenetic mutations capable of causing female infertility, including  and ZP3 . TP63 was not traditionally considered as a primary predictor in female infertility, later research demonstrated its involvement in regulating oocyte fate and fertility. An increasing number of reports have confirmed the role of p63 in the reproduction of mice and humans. Studies with mouse models revealed the expression of TAp63 isoforms in all female reproductive organs, with the highest concentrations in the vagina and ovaries [TP63-related cases were reported recently with infertility as a component of the syndromic phenotypes or isolated premature ovarian insufficiency (POI).Although  ovaries ,16. TP63Trp63 is integral for murine embryo cranio-facial, skin, and limb development [The roles of TAp63 and ∆Np63α in vivo have been previously reported by generating conditional knockout mouse models for either isoform, leading to distinct phenotypes. It has been demonstrated that elopment . A ∆Np63elopment . In a TAelopment ,46. Trp63 gene to knock out the function of p63α [Both ∆Np63 and TAp63 are expressed in the nuclei of male mouse testes and female mouse reproductive organs . The ful of p63α . Heterozygous female mice developed normal skin and thymus, two organs that canonically express p63α, but displayed ovarian dysfunction, atretic ovarian morphology, and follicle depletion. The ovarian phenotypes in these mice resembled those observed in humans with infertility. The oocyte loss observed in heterozygous ovaries was associated with the activity of TAp63β, which is rapidly degraded. The expression of TAp63β in this mouse model also triggered uncontrolled apoptotic death in primary oocytes independent of DNA-damage-driven phosphorylation. Previously reported p63 mutations related to human female fertility were also detected in this mouse model, lending credence to the hypotheses that the observed mechanism of fertility loss in mice could be similar to that in humans . AdditioTP63 mutations have been reported and classified into seven different diseases in the clinical setting, including five developmental disorders and two non-syndromic disorders. Point mutations in the DBD result in EEC  syndrome, which is characterized by limb deformation, cleft lip and palate, and ectodermal dysplasia [TP63 C-terminus are responsible for ankyloblepharon, ectodermal defects, and cleft lip/palate syndrome (AEC) [Human ysplasia . Mutatiome (AEC) . PatientLimb-mammary syndrome (LMS), acro-dermato-ungual-lacrimal-tooth syndrome (ADULT) and EEC can be summarized as ELA due to the overlap in patient presentation, including ectodermal dysplasia, limb defects, and cleft lip and palate. ELA mutations are mainly located in the DBD, implying the inhibition of DNA binding and transactivation in the C-terminus . Rapp HoTP63 [TP63 mutations in human patients reported in the literature are believed to not only affect epidermal development but also impact female fertility , which only affects limb development, and non-syndromic orofacial cleft (nsOFC), which causes cleft lip. The mutations associated with these non-syndromic disorders are usually found throughout the sequence of TP63 . TP63 muertility .TP63 gene [A 14-year-old female patient was described to have presented with all clinical LMS phenotypes as well as dysgenesis of the internal genitalia leading to infertility. Molecular analysis detected a 2-base pair (bp) deletion (c.1576-1577DelTT) in exon 13 of the P63 gene . This frTP63 gene. This mutation induces a modification of the reading frame and introduces a stop codon at base 1992 (TAA), 65 bp downstream to the canonic TGA stop. No other causes of premature menopause were identified in these patients. This reported mutation is located in the TID of the C-terminal region of p63, affecting the two α-isoforms of p63 (TAp63α and ∆Np63α) instead of the β- and γ-isoforms. Another family study reported three related female patients who presented with typical features of RHS/AEC and developed premature menopause around the age of 30 . A heterTP63: c.1927C>T, p.(Arg643*) mutation is located within the TID generating a truncated protein.Another case report described two adolescent sisters from an LMS family who presented with undetectable ovaries and hypoplastic uterine tissue, mammary glands, and nipples, which led to infertility . In contTP63 mutations and isolated POI. By analyzing 13 POI patients, the genetic cause of POI was identified in one patient who presented with primary amenorrhea and was diagnosed with isolated POI. The variant (c.1780C>T p.(Arg594Ter)) was identified to be a de novo nonsense variant in TP63 within the C-terminal exon and introduces a premature stop codon. The c.1780C>T p.(Arg594Ter) variant is located in the SAM domain and truncates TP63 before the TID. In addition to the above syndromic infertility cases, recent clinical studies have also reported a direct linkage between TP63 for variants. A distinct TP63 C-terminal truncating variant (c.1794G>A p.(Trp598Ter)) was detected in an unrelated patent who was diagnosed with isolated POI and primary amenorrhea with no additional causative variants. Other TP63 variant-related syndromic malformations were not detected in these two POI patients [The location of the c.1780C>T p.(Arg594Ter) variant results in nonsense-mediated decay in transcription and a truncated p63 protein. An additional 107 POI patients were recruited to further test the C-terminus of patients .TP63, which produced an aberrant transcript without disrupting the reading frame. This intragenic duplication was located in the DBD, resulting in steady aberrant TP63 mRNA in patients and, consequently, reduced p63 activity [One recent clinical study with 67 early POI patients described 37 ovary-related copy number variants (CNV) involving 44 genes. A 161 kilobase paternal intragenic duplication at 3q28 was identified in exons 2–10 of activity .TP63 have also been shown to decrease fertility for in vitro fertilization (IVF) patients [TP63 gene in IVF patients with advanced maternal age who were characterized to possess declined oocyte quality. This further confirmed the importance of p63 in maintaining oocyte quality and ovarian function.Mutations in patients . SelecteTP63-related ovarian insufficiency is diverse in patient presentations and includes isolated POI and syndromic POI with RHS, LMS, and AEC. Except for the intragenic duplication, all TP63 variants that have been associated with POI are located in the C-terminal region, which further confirms the involvement and importance of the TP63 C-terminus in regulating ovarian development and function. Thus, the relative contribution of the C-terminus in TAp63 to female fertility should be thoroughly considered. The current clinical literature reporting TP63 in patients appear to indicate a scientifically relevant relationship. On the other hand, there are potential undiscovered SAM domain interactors or post-translational modifications abolished in ΔNp63 [The wide expression pattern and high expression levels of p63 typically observed in female reproductive organs and the clinical manifestations associated with the mutations of in ΔNp63 , allowinFertility preservation for cancer survivors is also an unmet need. Ovarian insufficiency and infertility are major side effects of conventional chemotherapy and radiation therapy. TAp63 expressed in oocytes can be activated by traditional cytotoxic cancer therapies, leading to uncontrolled oocyte apoptosis and follicle pool depletion. Future research may elucidate the molecular mechanisms of germ cell death to repair lesions incurred during cancer therapy. This will contribute to generating and developing effective fertoprotective agents to preserve female fertility. While the expression of p63 has been reported in male germ cells, the contribution of p63 in spermatogenesis remains unclear. Further research on p63’s function and capacity in reproduction will boost our understanding of the molecular mechanisms of female/male infertility in addition to oncogenesis.


Robotic positioning systems are used in a variety of chemical instruments, primarily for liquid handling purposes, such as autosamplers from vials or well plates. Here, two approaches to the design of open-source autosampler positioning systems for use with 96-well plates are described and compared. The first system, a 3-axis design similar to many low-cost 3D printers that are available on the market, is constructed using an aluminum frame and stepper motors. The other system relies upon a series of 3D printed parts to achieve movement with a series of linker arms based on Selective Compliance Assembly Robot Arm (SCARA) design principles. Full printer design files, assembly instructions, software, and user directions are included for both samplers. The positioning precision of the 3-axis system is better than the SCARA mechanism due to finer motor control, albeit with a slightly higher cost of materials. Based on the improved precision of this approach, the 3-axis autosampler system was used to demonstrate the generation of a segmented flow droplet stream from adjacent wells within a 96-well plate. To identify which system provided finer movement control, a process adapted from ISO 9283:1998 3The files listed in Section 5.1.5.The file listed in The files listed in Section 6.2.The files listed in 4e.g., 100), so the listed prices are slightly higher than the exact component cost that is needed for construction. However, this approach provides a cost based on the list price that would be used for purchase.Additional tools that will be needed for the 3-axis autosampler system include:x_carriage_frame’ and ‘well_plate_holder’) were printed with 60% infill and 0.15 mm layer height using the same infill design.Power mitre saw (or similar), rotary cutting tool (or similar), hex key allen wrench set, screwdriver set, standard tap & die set, and a 3D printer. For this design, an Ultimaker 3 with Ultimaker PLA filament  was used. Most components were printed with a 100% infill and 0.1 mm layer height using the grid pattern infill design. Larger parts , paper towels, sandpaper, laser cutter, woodcutting saw, screwdriver set, wrench or pliers, and a soldering station, and a 3D printer. For this design, a Monoprice Mini V2 with Monoprice PLA filament  was used to print all parts with default settings: 22% infill, 0.1 mm layer height, and a grid pattern infill design.For both designs, the reported print settings were primarily based on default settings, so similar procedures on other 3D printers would likely be suitable to create the necessary parts.55.15.1.15.1.1.1Make the frame portions from the 6′ long piece of T-slotted framing extrusion (PN# 5537T101) by cutting 2 pieces to a length of 8.75″, 3 pieces to a length of 7.50″, and 2 pieces to a length of 6.50″. Cutting the T-slotted framing can be accomplished with a hacksaw or other hand tool, but this is one area where a square cut can make assembly easier. A power mitre saw is best, but if one is not accessible some vendors will make the cuts for a small fee.Make the Z-axis rails by cutting the ¼″ × 8″ long shaft (PN# 6061K101) into 2 pieces 3.65″ long. This can be accomplished with an angle grinder or rotary tool with a metal cutting wheel. A hacksaw or similar will not work as the shafting is very hard (60 HRC).Make the X, Y-axis rails by cutting the 8 mm shafting (PN# B07XD4FBVM) into 4 pieces 8.90″ long.Make the X, Y, Z-axis lead screws from the commercial parts . Two 9.25″ length pieces of B07QV4MRDD are needed for the X-axis, two 8.40″ length pieces of B07QV4MRDD are needed for the Y-axis, and two 3.40″ length pieces of B07C8P1DWX are needed for the Z-axis. The lead screws are softer material, and therefore they may be cut with an angle grinder, rotary cutting tool, or hack saw.Reduce the four X-axis slide bushings (PN# 6687K33) to 0.160″ thickness on one side to increase room for lead screw clearance . This pr5.1.1.21.well_plate_holder.STL with an appropriate tap ) and screw nut housings (leadscrew_nut_housing_*_axis.STL) by threading the indicated holes into each piece with an appropriate tap  by filing a small groove for the capillary into one piece. While both halves have a conical feature to aid insertion, it is advisable to only file one side as shown in Finish the capillary holder pieces (5.1.21.Press-fit the lead screw support bushings (PN# 6389K626) into the X, Y-axis lead screw supports . Secure 2.Bolt the X,Y-axis lead screw nuts (included with B07QV4MRDD) into their housings using four #6–32 × 0.5″ button head cap screws . The lea3.To attach the Y-axis carriage onto well plate holder , bolt fo4.To form the Z-axis carriage , press-f5.To prepare the X-axis carriage that also holds the Z-axis carriage, press-fit the Z-axis lead screw support bushing (PN# 6389K626) into the top of the X-axis carriage piece and secure it with bushing retaining button head cap screw #6–32 × 0.25″. Bolt the remaining X,Y-axis lead screw nut holder  to the c6.Combine the X-axis and Z-axis carriage components by inserting the two Z-axis slide rails (PN# 6061K101) through both components. Secure them in place with a #8–32 × 0.250″ button head cap screws for each rail on the bottom of the X-axis carriage piece. The portion of the Z-axis carriage designed to extend away from the lead screw should point toward the bottom of the combined piece .Fig. 14F7.To create the X,Y,Z-axis drive assemblies, attach the motor/lead screw couplings (PN# B073FDXHMG) to each of the three NEMA-17 axis drive motors, leaving approximately 0.325″ of the motor shaft open below the bottom of the coupling .Fig. 15D8.nema_17_xy_mount.STL and one of nema_17_z_mount.STL) using four #4–40 × 0.375″ button head cap screws for each plate  to connect the two 8.75″ pieces of 80/20 onto the two pieces prepared above, keeping the left aligned shaft supports on the left side. Attach two more angle brackets in a perpendicular position (1.180″ from the back of the base frame) that will be used to hold the frame bridge in place .Fig. 18T3.To assemble the frame bridge, prepare the two 6.50″ length pieces of 80/20 extruded aluminum with angle brackets, shaft supports, and the X-axis lead screw support at the positions shown in 4.Attach the frame bridge to the base frame using the angle brackets on the top of the base frame .Fig. 20CA clean, flat work surface makes frame assembly far easier. Calipers make an excellent layout instrument as they can be set to precise lengths and used to score lines directly on the framing. Framing / Fastener type: There are two distinct types of 20 mm square framing available: 80/20 and PZRT. We have selected 80/20 here as it is generally more available, but compatible fasteners tend to be more expensive. PZRT is more difficult to acquire in small lots, but there tends to be a wider selection of lower cost fasteners. Both formats offer standard and twist-in fasteners. For the purposes of this design, the M5 screws (PN# B07C9S7V1Z) and M5 flat nuts (PN# B01HKMF2EE) are used for standard connections. Twist-in fasteners are convenient as they can be placed into a section of framing even if the end is not open (to slide the fastener in). Whichever version is selected, the most important thing is to ensure that the frame and fasteners are compatible with each other.5.1.41.Install the Y-axis drive by inserting the two Y-axis slide rails through the shaft supports and the bushings on the Y-axis carriage (bottom of well plate holder) as shown in 2.Lean the auto-sampler onto its back side and thread the X-axis lead screw through its lead screw nut until it is fully inserted . Using t3.Repeat a similar process for the installation of Z-axis drive by threading the Z-axis lead screw through its lead screw nut already installed in the X-axis carriage frame . Then, h4.Inspect the completed 3-Axis Autosampler System construction.*Note: Simple modifications can be made with additional M5 screws and flat nuts, 3D printed mounts, and additional cuts of 80/20 extruded aluminum to mount electronic parts to the back of the frame or install a mount for a Raspberry Pi-compatible touchscreen interface on the front of the frame to further integrate components of the system.5.1.51.Make sure the supply is not plugged in, as the exposed wires can be dangerous. Ensure that the wires are properly secured into the barrel plug splitter (included with PN# B073QTNF9F), then connect them to the screw terminal on the RAMPS board (PN# B06XZ46PDJ) as shown in Insert two pieces of 18-gauge wire into the ends of a barrel plug splitter for eventual connection to the power supply. 2.With the power supply not plugged in, place each stepper motor driver (PN# B01FFGAKK8) into the RAMPS board . These d3.Connect the RAMPS board to the Raspberry Pi 3B GPIO pins using the pin diagram shown in 4.refV) for the stepper motor drivers, calculate an appropriate value based on the maximum current (maxI) for the motor using the following equation:To set the reference voltage  can be laser cut from a single sheet of ¼” thick plywood (PN# 958719), as shown in The first major piece of the SCARA autosampler to be constructed is the base. The three required 14″ × 9″ base pieces  on a flat, level surface. Place a layer of paper towel over the scrap board to avoid adhesion from glue leakage Place the bottom base piece (the one not containing an open square in the middle) on top of paper towel, with the corner containing a single hole placed at the bottom left. Cover the top surface with wood glue (PN# 107209) and spread it around with a paper towel or brush: the goal is to have a relatively thick, even layer of glue spread out over the top surface, except for the region for the cutout hole on the top two base pieces . Place t3.Place one of the two remaining boards on top of the glue layer, using the dowel pins for alignment. Press tightly down, cleaning up any glue that is squeezed out along the edges or into the well plate hole in the middle of the base. On top of this new piece, place another layer of glue, spread it out evenly, and place the final base piece board on top . Repeat the glue cleaning procedure along the edges and in the central recess. The full assembly is shown in 4.Place another paper towel on top of the base, followed by the remaining scrap piece of wood. Tighten 6–8 clamps around the stack , with th5.Begin the glue curing process, which may take up to 24 h depending on the selected adhesive. To clean excess glue from the base once curing is complete, a razor (or similar) blade or sandpaper can be used to remove glue that has been squeezed out of the edges. A drill  or other cutting tool can be used to remove excess glue from the holes that did not contain the dowel pins. A completed SCARA base after curing and cleaning is shown in 5.2.21.ParallelMountA_top.STL and ParallelMountB_top.STL, respectively), with the head of the screw placed into the recessed countersink hole so that it below the top surface of each plate top. Fit each plate front  and back  onto the screws: the thinner piece for the right and left servomotor modules should be on the side closer to the large hole cutout on each of the top plates. The rounded corners for all pieces will match when properly aligned. The slot cutout between the two holes for each front and back piece should be facing the center. Once the components are in the correct position, attach them by tightening a #4 nut onto each screw. The completed assemblies are shown in To assemble the left and right servomotor plates, insert four #4 × 0.75″ pan head screws into the four corners of each top piece  on top of the threads and tighten in place using two M3 nuts .Fig. 34F3.Place two more M3 × 20 mm screws through the holes shown on the bottom of 4.Align the combined left servomotor plate on top of the servomotor by placing the middle of the large hole over the servomotor spline and the four mounting holes on both the plate and motor/stand. Attach the plate to the motor and stand using four M4 × 20 mm screws tightened into the threaded holes on the stand. The completed component is shown in 5.Riser.STL) rather than directly onto the base . Two #4 × 0.5” screws are placed through the holes on the rectangular section that is orthogonal to the larger part of the plate. Align the screws with the threaded holes on the coil body, making sure that the plunger opening is facing the same direction as the top of the end effector  and the end effector effector . Tighten2.CapillaryRail.STL) and lever fulcrum (Fulcrum.STL) onto the top of the capillary plate using #4 × 0.5″ screws and #4 nuts in the positions shown in Attach the capillary rail (3.LeverAtt.STL) and the main lever piece (LeverMain.STL) so that the longer holes have a gap between them. Connect the two pieces through the smaller holes using a #4 × 0.5″ screw and #4 nut. Then, attach the solenoid plunger to the other side of the main lever piece using a M2 × 10 mm screw and M2 nut (Arrange the lever attachment (d M2 nut . One hol4.CapillaryClampA.STL and CapillaryClampB.STL) with #4 × 0.75″ screws so that they ride within the capillary rail. The capillary guide (CapillaryGuide.STL) is then attached to the bottom of the capillary plate with #4 × 0.75″ screws. The complete assembly is shown in To begin attaching the lever-plunger assembly to the capillary plate, cut the compression spring (PN# 9657 K107) to a length of approximately 0.75″, slide it over the plunger, and insert the pointed end of the plunger down into the solenoid coil body. Align the central lever hole with the lever pivot hole and connect with a #4 × 0.5″ screw. Place a drop of threadlocker (PN# 1810A27) onto the exposed threads of this screw, hand-thread the nut onto the screw, and let the threadlocker cure for 10 min before continuing. Do not fully tighten the nut, as that will restrict the motion of the lever. Attach the capillary clamps (5.LinkA1.STL) and B1 (LinkB1.STL), with the spline hole in the horn aligned with the larger hole on the link and the wings of the horn in line with the slots. Tighten two #0 × 0.5″ thread-forming screws through the link and into the horn so that the threads are on the same side as the horn. The screw for the inner slot can be placed in any of the holes of the horn wing. The screw for the outer slot should be placed in the hole closest to the spline. One of the horn-link connections is shown in LinkB2.STL) using a 0.2″ × 0.5″ Chicago screw, adding threadlocker to the threads before it is tightened. Let the connected pieces sit until the threadlocker sits. .To begin preparing the linkages that are connected to the capillary plate, attach the R-I  horns onto links A1 (6.Attach the combined B1/B2 link (right sub-linkage) to the hole on the right side of the capillary plate with a 0.2″ × 0.5″ Chicago screw .7.LinkP1.STL) and P2 (LinkP2.STL) to the elbow (Elbow.STL). It is important that the elbow be oriented correctly since it is not symmetric. With the longest flat edge up, the shortest flat edge should be on the right (Attach links P1 (he right . With th8.LinkA2.STL) to the remaining hole on the elbow (Connect links A1 and A2 (he elbow . Link A19.Attach the completed left sub-linkage to the capillary plate as shown in 10.Align the horn on the right sub-linkage (link B1) with the spline on the right servomotor and tighten with a spline screw .Fig. 49C11.Complete the linkage assembly by aligning the horn on the left sub-linkage (link A1) with the spline on the left servomotor and tightening with a spline screw. Then, connect the remaining hole on link P1 to the remaining hole on the left servomotor plate with a 0.2″ × 0.5″ Chicago screw. The completed linkage assembly is shown in 5.2.4Although the Raspberry Pi does not need to be connected to the base, four holes are open in the bottom right hand corner of the base. Four M2.5 × 15 mm screws can be oriented with the threads facing up through the bottom of the base and connected to four M2.5 × 10 mm standoffs. Once the standoffs are in place, position the four corners of the Raspberry Pi onto the standoffs and connect with M2.5 × 5 mm screws. The finished position of the Pi is shown in 5.2.5Supporting Information. The following steps can be performed once the boards have been completed.1.Plug the power supply board into the control board, as shown in 2.Plug the wires for the joystick and buttons into the joystick board and control board as shown in 3.Solder the ends of the solenoid wire to the solenoid and then connect the solenoid to the header on the control board as shown in 4.Connector 0 on the control board. The right servo should be connected to Connector 1. Ensure that the signal (yellow or white) wire is facing up when connected  and the column with the number (1–12). After pressing enter, the linkage will move to that position. The linkage will print out the x and y coordinates of the sampling head. Press the joystick down (button 1) to clear the entry and enter a new vial.2.Auto Mode – “a”: This mode will step through all of the vials on the microplate. It starts at A1 and steps through all columns before moving to the next row. It starts at column 1 for each row.3.Sequence Mode – “s”: The program will prompt for a sequence of vials to step through. The vials should be separated by commas in the format “A1, B2, C3, etc.” Press enter to start the sequence.4.Calibrate Mode – “c”: This mode calibrates the autosampler by moving to a set of points and having the user align the sampling head using the joystick. It will overwrite the existing calibration files  or create new files if they do not exist. The linkage will move near the first calibration point and ask the user to use the joystick. Move the sampling head directly over the vial and press the joystick (button 1) when aligned. Repeat this process for the remaining calibration points.5.Quit – “q”: Exits the program.The SCARA Autosampler offers several modes accessed through a command line interface . Run the77.1l  and recording the new position with a dial caliper, and finally returning to the “home position”. Measurements for the 3-axis design were made with calipers that provide 0.0005” resolution . This process was then repeated five more times, with accuracy calculated by:ā is the mean value of the final calculated position for all trials for a given axis. The repeatability was then calculated using the following series of equations:ia is the deviation for an individual trial. Bi-directional movements were used for this test (a slight deviation from ISO 9283) to account for the potential of leadscrew backlash in the measurement. For the SCARA autosampler, the same process was conducted, although the Z-axis was not tested as it only moves in two positions based on the solenoid control. Measurements for the SCARA design were made with a dial indicator that provides 0.001″ resolution . Results of the process are shown in To determine the motion characteristics of each movement design, a protocol based on ISO 9283 No issues were encountered during the development and routine operation of either platform described here, although no in-depth study on total performance lifetime was conducted. Due to the modular nature of these designs, any component that fails can be changed without the need to replace the entire system.7.2Figure S1. Signal was recorded as RGB intensity over time with ImageJ To demonstrate the use of the 3-axis autosampler for droplet formation, two adjacent wells of a 96-well plate were milled slightly below the planar surface, filled with green and red food dye , respectively, and then covered with a layer of perfluorodecalin (PFD) oil  up to the planar surface. A 44 cm segment of 100 µm inner diameter (i.d.), 360 µm outer diameter (o.d.) perfluoroalkoxy (PFA) tubing was coupled to a 250 µL gas-tight syringe  using a 1/16″ PEEK union with 1/16″ o.d. tubing sheaths used with both the syringe and the PFA tubing . To generate flow, a Chemyx Fusion 200 syringe pump  was operated in withdraw mode at a rate of 2.5 µL/min. The droplets were formed by moving the tube inlet between the oil layer and the two color wells in an alternating fashion. The motors were operated at 75 RPM with a 0 s delay within each well. The droplet stream was monitored on a stereo microscope (1-4x magnification) coupled to a Moticam 1080 HD camera . This video is included (at 2x playback speed) as 8This report details the design and construction of two mechanisms for movement control that can be used for chemical sampling: 3-axis stepper motors and SCARA-based servomotors. Both models provide adequate precision to move to individual wells with a 96-well plate, although higher precision was observed for the 3-axis design. The advantage of the SCARA design is reliance upon 3D printed parts, as it simplifies the construction compared to the need to modify commercial parts like in the 3-axis sampler. Once the relevant parts are obtained , both builds can be completed in approximately 3–4 h, and similar skill levels are required to construct each design. Based on their open-source design principles, both systems can be further adapted to suit more specific needs for robotic movement in chemical measurement systems (an increasing need in the field 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.


The contribution of UDP-glucuronosyltransferases (UGTs) enzymes to the metabolism of ticagrelor to its glucuronide conjugation, ticagrelor-O-glucuronide, in human liver microsomes (HLM) and human intestinal microsomes (HIM), was well characterized in the current study. The inhibition potential of human major UGTs by ticagrelor and ticagrelor-O-glucuronide was explored. The inhibitory effects of ticagrelor-O-glucuronide on cytochrome P450s (CYPs) enzymes were investigated as well. Ticagrelor glucuronidation exhibits substrate inhibition kinetics in both HLM and HIM with apparent Km values of 5.65 and 2.52 μM, Vmax values of 8.03 and 0.90 pmol min−1·mg protein−1, Ksi values of 1,343.0 and 292.9 respectively. The in vitro intrinsic clearances (Vmax/Km) for ticagrelor glucuronidation by HLM and HIM were 1.42 and 0.36 μl min−1·mg protein−1, respectively. Study with recombinant human UGTs suggested that multiple UGT isoforms including UGT1A9, UGT1A7, UGT1A3, UGT1A4, UGT1A1, UGT2B7 and UGT1A8 are involved in the conversion of ticagrelor to ticagrelor-O-glucuronide with UGT1A9 showing highest catalytic activity. The results were further supported by the inhibition studies on ticagrelor glucuronidation with typical UGT inhibitors in pooled HLM and HIM. Little or no inhibition of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7 by ticagrelor and ticagrelor-O-glucuronide was noted. Ticagrelor-O-glucuronide also exhibited limited inhibitory effects toward CYP2C8, CYP2D6 and CYP3A4. In contrast, ticagrelor-O-glucuronide weakly inhibited CYP2B6, CYP2C9 and CYP2C19 activity with apparent IC50 values of 45.0, 20.0 and 18.8 μM, respectively. The potential of ticagrelor-O-glucuronide to cause drug-drug interactions warrant further study.Ticagrelor is the first reversibly binding, direct-acting, oral P2Y Ticagrelor  and exogenous compounds  are largely attributed to the glucuronidation catalyzed by UGTs isoforms . CurrentThe current study was conducted to investigate the contribution of each UGT isoforms to the glucuronidation of ticagrelor in human liver and intestine using human liver microsomes (HLM), human intestinal microsomes (HIM), and recombinant UGTs. The inhibition potential of human major UGT by ticagrelor and ticagrelor-O-glucuronide , dextromethorphan (purity >95%), dextrorphan tartrate salt (purity >98%), and furafylline  were purchased from Toronto Research Chemicals . Reference standard of ticagrelor O-Glu (purity 96.5%) and trifluoperazine N-glucuronide (purity>96%) were purchased from TLC Pharmachem . Reference standards of atazanavir (purity 98%), celastrol (purity 98%), SN-38 (purity 99%), chenodeoxycholic acid (purity 98%), trifluoperazine (purity 99%), mefenamic acid (purity 98%), mycophenolic acid (purity >95%), 3′-azide-3′ deoxythymidine (purity 98%), hecogenin (purity 80%), alamethicin (purity >98%) and uridine 5′-diphosphoglucuronic acid trisodium salt  were purchased from J&K Scientific . N-Acetyl serotonin (purity 98%) and niflumic acid (purity >98%) was purchased from TCI Development Co., Ltd. . Testosterone (purity >98%) was purchased from European Pharmacopoeia. 6β-hydroxy testosterone (purity >99%) was purchased from Dr. Ehrenstorfer GmbH . Diclofenac (purity >98%) was purchased from Aladdin Industrial Corporation . Diazepam injection as internal standard was purchased from Tianjin Kingyork Pharm Co., Ltd. . Pooled HLMs  and pooled HIMs  were purchased from XenoTech LLC . Recombinant human UGTs Supersomes, such as UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, and UGT2B17 expressed by baculovirus-infected insect cells were purchased from BD Gentest . Formic acid was purchased from Sigma-Aldrich . Methanol and acetonitrile of mass spectrometry grade were purchased from Thermo Fisher Scientific . Purified water was prepared using a Milli-Q water purification system from Millipore Corporation . All other chemicals were of the highest purity commercially available.Reference standard of ticagrelor (purity 95.0%), 7-Ethyl-10-hydroxy-camptothecin (SN-38) glucuronide (purity 97%), chenodeoxycholic acid 24-acyl-β-D-glucuronide (purity 96%), N-Acetyl serotonin β-D-glucuronide (purity 97%), mycophenolic acid β-D-glucuronide (purity 96%), 3′-azide-3′-deoxythymidine β-D-glucuronide sodium salt (purity 97%), bupropion (purity 98%), hydroxy bupropion (purity >98%), amodiaquine (purity >95%), N-Desethyl amodiaquine hydrochloride (purity >95%), 4′-hydroxylation diclofenac (purity >95%), S-mephenytoin, Sin vitro incubation system by using HLM, HIM, and recombinant human UGTs. The incubation system consisted of enzyme sources , various concentrations of ticagrelor , alamethicin (50 μg/mg protein), 5 mM MgCl2, 100 mM tris-HCL buffer (pH 7.4), and 5 mM UDPGA in a total volume of 100 μl. Alamethicin is a pore-forming polypeptide used to activate UGTs in HLM and HIM  potential caused by ticagrelor and ticagrelor-O-Glu through the inhibition of the major human UGTs enzymes, selective UGT probe substrates were co-incubated with ticagrelor or ticagrelor-O-Glu in HLM. The formation of metabolites, SN-38 glucuronide, chenodeoxycholic acid 24-acyl-β-D-glucuronide, trifluoperazine N-glucuronide, N-Acetyl serotonin glucuronide, mycophenolic acid glucuronide and 3′-azide-3′-deoxythymidine β-D-glucuronide, which could reflect the enzyme activities of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, respectively, were quantified using LC-MS/MS. Three different concentrations of ticagrelor or ticagrelor-O-Glu  were added to the incubation system in the presence of selective UGT probe substrates , respectively. The assays were performed as described above. The control was treated with a vehicle solvent only. A triple determination was performed.m values: 150 μM bupropion, 1.26 μM amodiaquine, 10 μM diclofenac, 20 μM mephenytoin, 20 μM dextromethorphan and 80 μM testosterone (in house data)) in potassium phosphate buffer  was pre-incubated at 37°C for 5 min. Then, NADPH (10 mM) was added to initiate the reaction. The final volume of the incubation mixture was 100 μl. After incubation at 37°C for specific time points, the reaction was terminated by the addition of 200 μl of ice-cold methanol solution with internal standards (diazepam and furafylline). Preliminary screening experiments with selected concentrations of ticagrelor-O-Glu  were carried out to determine the inhibition potency. For those that produced approximately 50% inhibition of CYPs enzyme activities at a ticagrelor-O-Glu concentration between 1 and 100 μM, IC50 was further determined with various concentrations of ticagrelor-O-Glu . All the experiments were performed in triplicates. The supernatant was obtained after centrifugation at 14,000 rpm for 10 min and injected into LC-MS/MS for the analysis of the respective metabolites. The formation of respective metabolites was determined to reflect P450 isoform activities. The metabolites quantified for CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were hydroxy bupropion, N-Desethyl amodiaquine, 4′-hydroxylation diclofenac, S-4-hydroxy mephenytoin, dextrorphan and 6β-hydroxy testosterone, respectively.The potential inhibitory effects of ticagrelor-O-Glu on the activities of major CYPs, i.e., CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, were evaluated by co-incubation ticagrelor-O-Glu with selective P450 probe substrates in HLM. Each incubation mixture containing 0.2 mg/ml HLM, ticagrelor-O-Glu and respective substrate  in both the positive and negative electrospray ionization mode . The chromatographic separation was performed on a Kinetex C18 column  with the mobile phase consisting of purified water containing 1% formic acid (A) and methanol (B) with linear gradient elution. The linear gradient program was as follows: 0–1 min, 20% B; 1–5 min, 20–80% B; 5–7 min, 80% B; 7–8 min, 80–20% B; 8–9.5 min, 20% B. The autosampler temperature was maintained at 4°C. A methanol/acetonitrile/isopropanol  solution was used as the needle rinse solution. The detailed MRM acquisitions and mass spectrometry voltages for the fifteen analytes are listed in V is the velocity of the reaction, S is the substrate concentration, Km is the Michaelis-Menten constant, Vmax is the maximum velocity, and Ksi is the substrate inhibition constant. Intrinsic clearance (CLint) was calculated by dividing Vmax by Km.Kinetic analyses were performed using the mean values of triplicate determinations with either pooled HLM, HIM, or recombinant human UGTs. All data were analyzed by nonlinear regression analysis using the GraphPad Prism 6 . Michaelis-Menten kinetics  or subst50 values using the GraphPad Prism 6 .The percent inhibition relative to the control was calculated to indicate the UGTs or CYPs inhibition. For inhibition exceeding 50% of the control activity, nonlinear regression analysis was used to estimate the ICt-test. A p-value less than 0.05 was considered statistically significant.GraphPad Prism 6  was used for statistical analysis. The Kolmogorov–Smirnov test was used to check the normal distribution of continuous data. The statistical comparisons of percentage of control activity (%) between the control and experimental groups were carried out by a two-tailed m values of 5.65 and 2.52 μM, Vmax values of 8.03 and 0.90 pmol min−1 mg protein−1, Ksi values of 1,343 and 292.9 respectively. respectively. The in vitro intrinsic clearances (Vmax/Km) for ticagrelor glucuronidation by HLM and HIM were 1.42 and 0.36 μl min−1·mg protein−1, respectively  was used to screen the UGTs isoforms that may participate in the formation of ticagrelor-O-Glu. The results suggested that human UGT1A9, UGT1A3, UGT1A4, UGT2B7, UGT1A1, UGT1A7 and UGT1A8 are all involved in the metabolism of ticagrelor to its glucuronidation metabolite . In contTo confirm the results from recombinant enzymes, selective UGTs inhibitors were added to the incubation system individually for the comparison with the formation of ticagrelor glucuronidation in the absence of inhibitors. The results are shown in 50 values for these UGT isoforms were close to or greater than 100 μM  of ticagrelor after a 100 mg dose of b.i.d. in humans was 0.81 μg/ml (Cmax of ticagrelor. However, regarding metabolism based DDIs, the liver concentration should be used since it is closer to the concentration at the enzyme site. Although the exact concentration in the liver is difficult to predict, it can be estimated from the volume and flow rate of portal blood. In healthy adults, the volume of portal blood space is approximately 450 ml, and portal blood flow rate is ∼600 ml/min (in vivo. The results indicated that both ticagrelor and ticagrelor-O-Glu exhibited weak inhibition toward UGT1A6, UGT1A9 and UGT2B7. By contrast, ticagrelor also inhibited UGT1A1 and UGT1A3, which is not noted for ticagrelor-O-Glu. No significant inhibitory effect on UGT1A4 by neither ticagrelor nor ticagrelor-O-Glu at concentrations up to 100 μM. Thus, the DDI risk attributed to the inhibition of these UGT isoforms by ticagrelor and or ticagrelor-O-Glu is low. Regarding the inhibition of CYPs, selected concentrations of ticagrelor-O-Glu were used to do the preliminary screen. Although significant inhibition toward CYP2C8, CYP2D6 and CYP3A4 by ticagrelor-O-Glu was noted when the concentration of ticagrelor-O-Glu is as high as 100 μM, the inhibition potencies were not so strong that IC50 values for these isoforms by ticagrelor-O-Glu were not further determined. However, ticagrelor-O-Glu weakly inhibited CYP2B6, CYP2C9 and CYP2C19 activities with apparent IC50 values of 45.0, 20.0 and 18.8 μM, respectively.For UGT inhibition, the concentration range of ticagrelor used in this study was 0.52–52.26 μg/ml. In contrast, the steady-state maximal plasma concentrations (81 μg/ml . It seem0 ml/min . Therefoin vivo, it is difficult to evaluate its role in DDI risk in clinic. Even so, it is important to note that although the relatively low concentrations of ticagrelor-O-Glu in comparison to its major active metabolite, AR-C124910XX, the potential of ticagrelor-O-Glu to cause DDIs should also not be ignored. Further study is warranted to reveal the in vivo exposure of ticagrelor-O-Glu and investigate the DDI risks caused by ticagrelor-O-Glu as inhibitors of CYP2B6 and CYP2C19.Metabolites-mediated DDI attributed to the inhibition of cytochrome P450 enzymes by glucuronide metabolites are relatively common in clinical scenarios . The stu50 values of 45.0, 20.0 and 18.8 μM, respectively. When ticagrelor is co-administered with CYP2B6, CYP2C9 and CYP2C19 substrate drugs in clinic, the possibility of clinically significant DDI deserves further study.In summary, the present results indicate that ticagrelor glucuronidation is mainly catalyzed by UGT1A9 in human liver, followed by UGT1A7, UGT1A3, UGT1A4, UGT1A1, UGT2B7, and UGT1A8 in human liver and/or intestine. Little or no inhibition of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7 by ticagrelor and ticagrelor-O-glucuronide was noted. Ticagrelor-O-Glu also exhibited limited inhibitory effects toward CYP2C8, CYP2D6 and CYP3A4. In contrast, ticagrelor-O-glucuronide weakly inhibited CYP2B6, CYP2C9 and CYP2C19 activity with apparent IC


Numerical simulations of glacier slip over diverse hard beds indicate Coulomb slip behavior like that of deformable beds. Ice-sheet responses to climate warming and associated sea-level rise depend sensitively on the form of the slip law that relates drag at the beds of glaciers to their slip velocity and basal water pressure. Process-based models of glacier slip over idealized, hard (rigid) beds with water-filled cavities yield slip laws in which drag decreases with increasing slip velocity or water pressure (rate-weakening drag). We present results of a process-based, three-dimensional model of glacier slip applied to measured bed topographies. We find that consideration of actual glacier beds eliminates or makes insignificant rate-weakening drag, thereby uniting process-based models of slip with some ice-sheet model parameterizations. Computed slip laws have the same form as those indicated by experiments with ice dragged over deformable till, the other common bed condition. Thus, these results may point to a universal slip law that would simplify and improve estimations of glacier discharges to the oceans. Major contributions of ice discharge to the oceans that result largely from rapid slip of glaciers over their beds are accelerating mass losses from ice sheets and associated sea-level rise  beds, the ratio, τb/N, cannot exceed the maximum slope, mmax, of the up-glacier (stoss) sides of bed bumps .N would decrease resistance to slip, promoting faster flow and glacier instability, including glacier surge propagation  or photogrammetry from an unmanned aerial vehicle (UAV) . Proglacial areas selected for study, in Alberta, Canada, and in Switzerland (see figs. S1 and S2), consist of well-exposed sedimentary, metamorphic, and igneous bedrock with contrasting patterns of discontinuities. Associated bed morphologies range from meter-scale steps with planar stoss treads trending sub-perpendicular to the glacier-slip direction to bumps with convex stoss surfaces streamlined obliquely to the slip direction .2, but because modeling glacier slip over 3D beds is computationally intensive, we develop sliding laws over smaller areas (140 to 270 m2). These areas enclose subsections of the bed, which we call representative elementary areas (REAs), chosen to be morphologically representative of the full areas surveyed. REAs are identified using nine morphological characteristics that potentially affect sliding physics, and principal component analysis (PCA) is used to score the importance of each characteristic (see fig. S3). Scores for the full areas surveyed are compared to those for subsections of the bed to isolate REAs  . We determine the slip velocity, ub, in each case by integrating the velocity at the sole of the ice layer in the down-glacier direction. Resultant slip laws are presented as scaled, dimensionless drag relations   under a N is varied. Owing to the irregular morphologies of the REAs, which preclude scaling As to a simple geometric attribute of the bed, the value of As is computed through application of the model at velocities too small to induce cavity growth.This scaling of slip velocity allows comparison among the REAs of differences in drag relations with and without cavities and results in a relation that is independent of whether the velocity or b/N increases toward a bounding value with increasing scaled slip velocity . The general forms of the slip relations are not sensitive to domain-edge tapering (fig. S7).Conspicuously lacking from the derived drag relations  is the svelocity . For a sbehavior . The Coubehavior  but alsommax, controls drag for the case of idealized beds .lculated  if largen = 3  for the four proglacial areas are constructed using data from a TLS and photogrammetry techniques with images collected from a UAV (fig. S1). TLS data collected with a Riegl VA-400 scanner are used for the proglacial area of Castleguard Glacier. Scan positions are selected to assure scan overlap and mitigate shadow effects from bedrock features. The resulting point-cloud data are merged, filtered using reflectance  and deviation  attributes, and manually cleared of spurious points with the RiSCAN Pro software package .To determine an REA for each proglacial area, morphologic attributes of 10 m × 10 m subsections of a DEM are compared with those of the entire proglacial area. The DEMs are trimmed to a square and then subdivided into sections using a sliding window with a 2-m step spacing. The subsections are detrended, and their morphologies are characterized after Leach . For all other parameters, Xν is set to the mean of the parameter set. The Xν values are normalized with the other parameter values to a mean of zero and variance of one before performing the PCA, owing to the different units of parameters. The PC scores (magnitude of each PC in the PC coordinate system) are estimated for the normalized parameter values of individual subsections, γiν,, and the representative parameter values of the entire proglacial area, γν, where i denotes the subsection index. The representative PC scores, γν, are subtracted from each subsection’s score, γiν,, resulting in a similarity index, θi, for each subsection that is used to find the most representative subsection of the DEMTo find the REA of a proglacial area, PCA  with combined similarity indices closest to zero (greatest similarity) as a set of potential REAs. Each subsection of this set is expanded by 5 m in each direction using the surrounding bedrock topography, resulting in a 20 m × 20 m section centered on the statistically representative 10 m × 10 m subsection. As a measure of periodicity of any given rectangular section, we consider the To make the boundaries of a selected rectangular section fully periodic, we use a periodicizing tapering algorithm with a taper width of 1 m at the boundaries (see text S2). A 2D rectangular sliding window is used to average the elevation values within the tapered zone. The maximum size of the window is 2 m in each direction, with the window centered on the point  that is averaged. The size of the window progressively decreases to zero as averaged coordinate points approach the inner edge of the tapered zone (see fig. S8). The varying size of the averaging window results in a smooth transition from the tapered part of the section to its untapered interior. In choosing a modeling domain, for the periodicity measure, we considered rectangular sections only large enough so that the centered REA is not affected by the tapering algorithm. For the four proglacial areas, comparing the PC scores of the tapered and low-pass–filtered rectangular section containing the selected REA to the corresponding unmodified section indicates that only a small statistical change in bed morphology is introduced by the tapering procedure. The relative change in the PC scores for the selected rectangular sections of the proglacial areas of Schwarzburg, Rhône, Castleguard, and Tsanfleuron are 0.07, 16.44, 1.31, and 0.98%, respectively. The high relative change of the Rhône proglacial area is due to the long wavelengths of its bed morphology and the small area of the selected rectangular section.zs, are given by the Stokes equations (5) and kinematic free-surface equation (7). For the model, ice occupies a Cartesian domain in a boundary layer at the glacier bed, which consists of the rectangular domain that includes the REA and surrounding tapered regions. To the top of the domain, we apply a flow velocity, ue, and ice overburden pressure, pi. Gravity in the thin boundary layer can be neglected. At the bottom of the domain, the glacier sole either is in contact with the underlying bedrock, zb, or is the roof of a water-filled cavity, zc. The bedrock is taken to be impenetrable and the water pressure in cavities, pw, to be spatially uniform over the bed. Water in a thin film divides ice at its pressure-melting temperature from rock where water-filled cavities are absent, so the low viscosity of water results in negligible tangential stresses everywhere at the glacier sole (free slip). Basal melting and regelation are neglected. At the vertical boundaries of the tapered domain, periodic boundary conditions apply, so ice deforms during slip over morphologically repetitive bedrock of infinite extent.The equations governing the viscous deformation of ice and steady-state geometry of the glacier sole, u = , and pressure, p, are then given as the solution ton and t are the outward-pointing normal and tangent to the boundary, respectively. The deviatoric stresses are related to strain rates through the constitutive equation for ice deformation  base mesh in 30 layers in the vertical (z) direction, resulting in prismatic/wedge elements. The same vertical mesh extrusion is used for all glacier topographies, with the density of element layers highest near the base of the computational domain. The top of the domain is fixed at z = 15 m, whereas the bottom of the domain adjusts to the geometry of zs in each time step. All base meshes are generated with a characteristic cell size (triangle edge length) of 0.15 m using the frontal Delaunay algorithm through the software Gmsh (et al. (The set of


Through interacting with Gαi/o‐coupled A1 receptors, the neuromodulator adenosine modulates a variety of cellular and synaptic activities. To determine the linkage from A1 receptors to a key intracellular signaling pathway, we investigated the impact of blocking A1 receptors on a subfamily of nonreceptor tyrosine kinases, that is, the Src family kinase (SFK), in different rat brain regions in vivo. We found that pharmacological blockade of A1 receptors by a single systemic injection of the A1 selective antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) induced an increase in autophosphorylation of SFKs at a consensus activation site, tyrosine 416 (Y416), in the two subdivisions of the striatum, the caudate putamen and nucleus accumbens. DPCPX also increased SFK Y416 phosphorylation in the medial prefrontal cortex (mPFC) but not the hippocampus. The DPCPX‐induced Y416 phosphorylation was time dependent and reversible. In immunopurified Fyn and Src proteins from the striatum, DPCPX elevated SFK Y416 phosphorylation and tyrosine kinase activity in Fyn but not in Src proteins. In the mPFC, DPCPX enhanced Y416 phosphorylation and tyrosine kinase activity in both Fyn and Src immunoprecipitates. DPCPX had no effect on expression of total Fyn and Src proteins in the striatum, mPFC, and hippocampus. These results demonstrate a tonic inhibitory linkage from A1 receptors to SFKs in the striatum and mPFC. Blocking this inhibitory tone could significantly enhance constitutive SFK Y416 phosphorylation in the rat brain in a region‐ and time‐dependent manner.Adenosine A Pharmacological blockade of adenosine A1 receptors enhanced phosphorylation and enzymatic activity of the Src family kinases such as Fyn and/or Src in the adult rat brain in a region‐ and time‐dependent manner. This supports an existence of a tonic inhibitory linkage from A1 receptors to a key and Fyn/Src‐dependent intracellular signaling pathway in neurons in vivo under normal conditions. In this study, we found that pharmacological blockade of A1 receptors enhanced phosphorylation and enzymatic activity of the Src family kinases such as Fyn and/or Src in the adult rat brain in a region‐ and time‐dependent manner. This supports an existence of a tonic inhibitory linkage from A1 receptors to a key and Fyn/Src‐dependent intracellular signaling pathway in the rat forebrain in vivo under normal conditions.Adenosine is an essential modulator in the central nervous system. Through interactions with G1, A2A, A2B, and A3) have been identified to date  activity, whereas activation of A2A and A2B receptors produces the opposite effect  coimmunoprecipitated in co‐transfected fibroblast cells  is a family of nonreceptor tyrosine kinases  and NAc. Other brain regions were also surveyed, which include the medial prefrontal cortex (mPFC) and hippocampus.We thus initiated the present study to investigate the possible linkage between A22.1The study was not pre‐registered. Adult male Wistar rats  that were purchased from Charles River  were kept at a facility  with water and food available ad libitum. Animals were housed two rats per cage and were used 5–6 days after habituation. The animal use and care protocol was approved by the Institutional Animal Care and Use Committee of University of Missouri‐Kansas City (protocol #: 1006).2.21 antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) on SFK Y416 phosphorylation in a total of three groups (n = 4 per group). DPCPX is selective for A1 receptors as it was approximately 1000‐fold more potent at A1 than A2A receptors in the striatum . Changes in Y416 phosphorylation were analyzed in the CPu, NAc, and mPFC. Third, we attempted to identify the members of SFKs that were regulated by A1 receptor signaling in two groups of rats (n = 4 per group). To this end, we analyzed changes in Y416 phosphorylation in immunopurified Fyn and Src proteins from the striatum and mPFC after vehicle or DPCPX administration . Finally, tyrosine kinase activity of immunopurified Fyn and Src was assayed in two groups of rats (n = 5 per group). Changes in Fyn and Src kinase activity in response to vehicle or DPCPX  were monitored in the rat striatum and mPFC. Doses of DPCPX (0.25 and 2.5 mg/kg) were chosen based on previous findings that an i.p. injection of DPCPX at 3 mg/kg blocked the anxiolytic‐like effect induced by the A1 selective agonist and positive allosteric modulator  and determined sample size by the sample size calculation . Sample size was not different between the beginning and end of the experiments. Inclusion/exclusion criteria were based on the health state of animals. We used an intraperitoneal (i.p.) injection to administer drugs. Drugs were calculated as the salt for their doses and were injected in a volume of 1 ml/kg. Age‐matched rats received a vehicle injection and served as controls. We first examined effects of the A2.32EDTA, 1 mM EGTA, 1% NP‐40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β‐glycerophosphate, 1 mM Na3VO4, and 1 μg/ml leupeptin . Samples were centrifuged at 800 g (10 min) and the supernatant was collected. After protein concentrations were determined, samples were used for immunoblots. Western blot was performed as described previously  and decapitated. Brains were removed and coronal slices were cut. The brain regions of interest  were dissected. In addition, the mPFC was dissected, which includes the anterior cingulate, prelimbic, and infralimbic cortex  was used to assess SFK kinase activity as described previously  with a multiple comparison post hoc test. The level of statistical significance was determined by a P value less than 0.05. Test for outliers was not performed. No rats were excluded from the analysis.After the normality of data was tested, we utilized GraphPad Prism 6  to analyze data. We used a two‐tailed unpaired Student's 33.11 receptors with DPCPX has any impact on SFK Y416 phosphorylation. To this end, we subjected rats to a single i.p. injection of vehicle or DPCPX at either 0.25 or 2.5 mg/kg. Rats were then sacrificed 20 min after DPCPX injection. Changes in Y416 phosphorylation were analyzed in different brain regions using western blots. We found that DPCPX at 0.25 mg/kg did not alter pY416 levels in the CPu  and were sacrificed at three different time points  after drug injection. Changes in pY416 levels in three brain regions  were assayed using western blots. In the CPu, DPCPX induced a significant increase in pY416 levels at an early time point , rats were sacrificed 20–30 min after drug injection. Fyn and Src proteins were immunoprecipitated from striatal and mPFC lysates. Phosphorylation at Y416 was then analyzed in Fyn and Src immunoprecipitates using western blots. In the striatum containing the CPu and NAc, a significant increase in pY416 levels was shown in immunopurified Fyn proteins in DPCPX‐treated rats compared to vehicle‐treated rats  of SFKs that respond to the As Figure . The pY4s Figure . In the s Figure  and D. T3.4Given an elevated response of Y416 phosphorylation to DPCPX, SFK tyrosine kinase activity is reasoned to be increased by the antagonist. To determine this, rats were treated with DPCPX . They were then sacrificed and striatal and mPFC lysates were prepared. Fyn and Src proteins were immunoprecipitated from these lysates. Kinase activity levels of immunopurified Fyn and Src were assayed. As shown in Figure 41 receptors in the regulation of SFKs in the rat brain in vivo. We found that a systemic injection of the A1 antagonist DPCPX at a higher dose (2.5 mg/kg) but not a lower dose (0.25 mg/kg) induced a substantial increase in the autophosphorylation of SFKs at Y416 in the two subdivisions of the striatum (CPu and NAc). DPCPX also increased Y416 phosphorylation in the mPFC but not the hippocampus. The increase in Y416 phosphorylation was rapid and reversible as revealed in a time course study. The DPCPX‐elevated Y416 phosphorylation occurred in immunopurified Fyn but not Src proteins from the striatum. In the mPFC, both Fyn and Src precipitates showed an increase in Y416 phosphorylation in response to DPCPX. Finally, a similar pattern of responses of Fyn and Src in their tyrosine kinase activity was observed in the striatum and mPFC after DPCPX administration. Our results demonstrate a significant response of SFKs to changing A1 receptor signals. A1 receptors seemingly exert an inhibitory regulation of Fyn and/or Src phosphorylation and activity in the striatum and mPFC under normal conditions.This study investigated the role of adenosine A1 receptors exerts a significant tonic modulation of functions of striatal neurons. For instance, an early study shows that a single dose of DPCPX and the nonselective adenosine receptor antagonist caffeine increased expression of the immediate early genes  in the striatum at both mRNA and protein levels ‐cyclopentyladenosine significantly altered Thr34 phosphorylation of dopamine‐ and cAMP‐regulated phosphoprotein of 32 kDa (DARPP‐32) in the striatum  underlying the effect of DPCPX on Fyn phosphorylation in the striatum. Of note, A1 receptors are also expressed in peripheral tissues such as cardiac and vascular cells (Headrick et al., 1 receptor agent injected i.p. could affect central Fyn phosphorylation indirectly via its effect on peripheral cardiovascular activities.Precise mechanisms underlying the effect of DPCPX on Fyn in the striatum are unclear. Postsynaptically, A1 receptors are negatively coupled to Src in the mPFC. Of note, in contrast to this negative connection, A1 receptors could positively modulate Src in different models. For instance, an A1 receptor agonist activated Src to form a signaling pathway linking A1 receptors to activation of nuclear factor kappaB in HEK293 cells (A. M. F. Liu & Wong, 1 receptor antagonist CPT reduced Src Y416 phosphorylation in acutely prepared mouse cortical slices containing somatosensory cortex (Deng et al., The expression level of Fyn is much greater than Src in the striatum (Pascoli et al., 1 receptors in SFK phosphorylation are poorly understood at present, it is imperative to investigate the response of SFK phosphorylation to adenosine receptor agents in female animals in future studies.Our studies on SFK phosphorylation were conducted in male rats. While sex differences in the role of AThe authors declare that there is no potential conflict of interest.https://publons.com/publon/10.1002/brb3.2254.The peer review history for this article is available at


Alpine cold ice caps are sensitive indicators of local climate. The adequate interpretation of this information in an ice core requires detailed in situ glaciological and meteorological records, of which there are few. The Weißseespitze summit ice cap (3499 m) presents an ideal case to compare past and present climate and mass balance, with limited ice flow, but close to 6000 years locked into about 10 m of ice. First-ever meteorological observations at the ice dome have revealed that over 3 years of observation most of the accumulation took place between October and December and from April to June. In the colder winter months, between January and March, wind erosion prevents accumulation. Melt occurred between June and September, ice was only affected during short periods, mainly in August, which caused ice losses of up to 0.6 m . Historical data points at a loss of of 34.9 ± 10.0 m between 1893 and 2018 and almost balanced conditions between 1893 and 1914. The local evidence of ice loss lays the basis for the interpretation of past gaps in the ice core records as past warm/melt events. The current Central European glacier retreat is historically unprecedented2 and results in large-scale down-wasting3. Therefore, records of past and present glacier state and fate are important, not only for science but also for society. Current monitoring is designed to cope with the role of glaciers as essential indicators of past climate conditions4. In the European Alps, records of glacier variability go far back5 and thus offer the most direct link between instrumental data and glacier area or mass balance as proxy records of climate. Climate information from Alpine glaciers includes past terminus positions recorded in the positions of moraines deposited during past glacier advances6, the structure and age of glacial deposits7, measured length changes as well as total and point mass balances. The timing and extent of glacier advances is greatly influenced by topography and dynamics8, and so are proxy data of terminus positions.Glaciers are icons and indicators of climate change and mitigation efforts9 to polar ice cores as cornerstone archives of past climate and atmospheric composition10. With the deposition of snow, additional properties of the atmospheric composition are archived . In the absence of prolonged mass loss, a chemical and structural stratification of ice exists which makes up the paleoclimatic record that can be accessed by drilling ice cores. However, ice core records from the polar regions are not necessarily representative for middle and low latitudes, especially for short-lived atmospheric constituents.The role of glaciers as climate archives is unique. It ranges from establishing the idea of a changing climate11. In the European Alps, ice core research aimed at continuous stratigraphic climate records focused on a few suitable drilling sites in the Western Alps. There, in locations above 4000 m, the low infiltration depth of (refreezing) meltwater, if it occurs at all, allows the preservation of the original layering of the accumulation12.At least for the Holocene period, important complementary information can come from ice cores drilled at cold (non-temperate) glaciers and ice caps of high mountain ranges in nonpolar areas15, sparking new efforts to retrieve this valuable paleoclimatic information which is currently threatened by warming17. In view of the predominant mass loss even at the summits of such sites, it is paramount to also take into account the role of surface mass balance in the interpretation of the associated ice core records. This opens up a new field of research questions, linking mass and energy balance consideration and modelling efforts to aid the interpretation of ice core records17.Recent studies have shown that also in the Eastern Alps, and at elevations below 4000 m, millennial-old ice and associated climate records may be preserved18, supported by numerical modelling17. As the age-depth profile of ice cores is not only relevant for paleo-hydrology and glaciology, but also for the interpretation of ice core records, this becomes a field of major significance.Direct mass balance measurements on summit glaciers complement paleo-mass balances derived from ice cores20. Strategies employing reference surfaces22 and point mass balances23 aim to discount the topographical and dynamical components.Geodetic or direct mass balances of glaciers include a feedback from glacier geometry. Several authors have presented methods to estimate the influence of those geometrical feedbacks, such as24. For the Austrian Alps, single high elevation stakes have been in operation, e.g. at Kesselwandferner glacier  from 1968 to 1983 and at Übergossene Alm (2850 m) from 1965 to 197526. Thickness records at least are available from repeat cartographic campaigns, where the summit elevation was often measured directly, e.g. for the Weißseespitze summit (3531 m anno 1922) at Gepatschferner glacier27.The logical consequence of such attempts is studying point mass balances at cold summit glaciers, which are frozen to the ground and thus almost stagnant. So far, only very few summit stakes have been installed within traditional and long-term mass balance programs. During the 1960s to 1980s, when most of today’s benchmark glacier in situ programs started, it took immense efforts to dig out and replace the stakes, so numbers were kept to a minimum. These point mass balance data sets, often unpublished and difficult to find, have already been compiled for the Swiss Alps30. Although high-elevation glaciers, especially in the Western Alps, were remarkably untouched by global warming31, englacial warming has been recorded despite insignificant volume changes32. Glacier outlet floods of meltwater trapped by still cold glacier parts also threaten, as happened at the Tête Rousse glacier cavity33.In the Western Alps, several prominent glaciers in summit locations have been investigated in detail, for example in the Monte Rosa and Mont Blanc regionThe present paper analyses present mass balance and more than a century of thickness change evidence as a basis for the detailed interpretation of the stratigraphy of the ice core drilled at Weißseespitze  between November 2017 and February 2021 Fig. . Snow heGlacier mass balance measurements at Weißseespitze summit comprise automatic measurements of snow height and ice thickness loss in subdaily resolution at the AWS and stake measurements in subseasonal resolution. During the 3 years of observation, snow height maximum values appeared in May and June, with an all out maximum snow depth of 2.4 m recorded in May 2018. During winter 2019/2020, snow height was around 0.5 m until May and reached its maximum with 2.2 m in June. Winter 2020/2021 presents a similar pattern, with less than 0.5 m snow height until February.Continuous records of ice ablation indicate the duration of the ablation season and the magnitude of ice melt at the AWS. Data gaps cause an underestimation of the seasonal ablation values measured, with 0.11 m in 2018 and 0.29 m in 2019. The length of the ablation season at the weather station was corrected by time-lapse camera images. No ablation was recorded at the AWS during summer 2020.At the ablation stakes, the spatial pattern of ice ablation is highly variable between years depending on the weather conditions and snow accumulation patterns Fig. , as evidThe Weißseespitze summit forms a glacier dome with circular contour lines from about 3470–3500 m Fig. . A bergs6 m3  is available only as monthly means/sums for 1800–2014  and August 2018 (1.9 °C) and close to 0 °C in September 2018 (− 0.4 °C), as well as in July 2019  higher than the average of the last 6 millennia and (ii) past similar events must have led to gaps in the ice core stratigraphy. The visual stratigraphy of the Weißseespitze ice core already shows a number of layers that suggest discontinuities, such as alternating sections of inclined and horizontal layers and a visible dark band  according to the HISTALP data . This is confirmed by the observed maximum snow heights of 2.43 m, assuming an average density of 400 kg/m48. It revealed 14 annual layers of firn within the upper 19 m, which were attributed to the accumulation of the years 1963 to 1949, reaching a density of 820 kg/m3. In the firn pit, several bands of superimposed ice were found, and water was observed to penetrate and saturate the firn layer. This is not the case at the WSS summit today, as no more firn is present there. So far, no supraglacial runoff system has been observed that would provide geomorphological evidence for melt water flowing on or in the ice body, water storage and penetration is limited to microcracks.The presence of continuous winter accumulation at WSS is also supported by data from a 20 m deep firn pit on Kesselwandferner at an altitude of 3240 m15. New approaches and dating techniques49 may soon allow determining the surface age at ice core sites with present negative mass balance, a problem that has recently become more widely recognized41.The comparison of the thickness loss at WSS to the age constraint of the firn pit on Kesselwandferner supports an age prior to the atom bomb tests consistent with the lack of tritium in the ice core. The thickness loss between 1969 and 2018 at the summit of Weißseespitze is similar to the depth of the snow pit at Kesselwandferner. Hypothesizing that basically no new layers formed during this period, the loss would therefore expose a surface from about 1950. Taking into account that the summer of 1947 was reported to cause a similar ice loss to 2003, today’s surface likely dates from layers formed prior to 1947. This is supported by the absence of the tritium peak from atmospheric nuclear bomb tests in the 1960s in the ice coreThe synopsis of in situ mass balance and the related processes, such as wind erosion and ablation, confirms that Weißseespitze summit ice cap is indeed sensitive to climate change. With hardly any temporal variability in accumulation for the last 200 years, the ice archive is very sensitive to warm summer temperatures. Today just a few days of ice melt can trigger negative mass balances with a complete loss of the annual accumulation. The thickness change of the summit ice cap could be constrained to 34.9 ± 10.0 m between 1893 and 2018 by analysing historical maps. This result was confirmed by historical photographs. The present annual melt rates of up to 0.95 m/year measured at single stakes are clearly higher than the average annual mean thickness loss of 0.28 ± 0.08 m/year for 1893 and 2018.50. However, in an Alpine summit glacier like Weißseespitze, with a mass balance regime highly sensitive to climate change, this link is far more complex. Reconstructions of Holocene temperatures42 and glaciation51 point to a fairly pronounced Holocene maximum temperature right before the reglaciation at Weißseespitze started, according to the age constraint of the basal ice layer of (5.9 ± 0.7) ka cal . There, mean annual horizontal flow velocity was 0.6 m/year, with a maximum of 0.7 m. Flow velocities at stake L2 at an altitude of 3296.8 m in 1968 are higher, with an average of 1.7 m/year and a maximum of 2.8 m/year. For both stakes, no local measurements of snow and firn density are available. Both records were the highest stake measurements in the Austrian Alps before the WSS stakes came into operation, and serve only as comparison and validation of the elevation changes recorded in the historical maps.An AWS was installed in the summit region at an elevation of 3499 m  profiles and stakes. The DGPS raw data, recorded per second, was corrected and adjusted in post-processing with the four closest STPOS (South Tyrolean Reference Station Service) base stations Mals-Malles, Meran-Merano, Bozen-Bolzano and Sterzing-Vipiteno. The achieved vertical and horizontal accuracy of the processed positioning data is within a range of ± 0.5 m. The synchronization of the GPR trace number with the DGPS data was done via the GPS time under consideration of the current 18 leap seconds to the GSSI-recorded UTC time.55.The ice thickness at the Weißseespitze summit was measured with GPR on 8 June 2017. Because of the expected low ice thicknesses, a GSSI 3102A antenna with a central frequency of 500 MHz was used. The radar sections were recorded at the uppermost area of the summit along several cross profiles Fig.  at an in55. The bedrock elevation is consistent with the ice core drillings. The vertical accuracy of the DGPS-measured surface elevation is ± 0.5 m, so that the accuracy of the bedrock elevation is constrained by surface elevation uncertainty and the ice thickness uncertainty of ± 10%.The bedrock topography of the summit was calculated from the GPR measurements and a bilinear interpolation between the bedrock interpretation of the GPR profiles, the glacier outlines as zero ice thickness and taking into account the surrounding ice-free topography from the 2018 DEM56. From 2006 onwards, the DEMs are based on high-resolution LiDAR data with 1 × 1 m pixel size. For the DEM of 2006, the uncertainty is ± 0.1 m56. The standard deviation at control areas for the DEM of 2018 was 0.032 m57. The effects of a seasonal snow cover, to be added to the nominal accuracy of the DEMs, can be estimated by the maximum snow cover recorded at the AWS to be about 2 m.Basic data for the calculation of past elevation and volume changes are provided by different available DEMs as given in Table The DoDs (DEM of Difference) were calculated in ArcMap with the “minus” tool and took into account the basic resolution of the data and exact overlapping raster cells. The volume change of the summit ice cap was calculated for the area where the bedrock is above the 3470 m.a.s.l. contour line by summing up the volume changes in each pixel of the DoD grids.58, an uncertainty of ± 1–2 m at the glacier tongue, where tacheometry was applied, and an uncertainty of ± 0.10 m at photogrammetrically processed upper parts of the glacier. For our analysis, we used maps where tacheometric surveys are indicated by cartographic symbols.For the time before 1969, elevation and shape of the ice cap at Weißseespitze are recorded in historical maps and a number of photographs. Although this summit has been more remote and less prominent than others , the Gepatschferner glacier has been subject to early glaciological surveys. The summit of Weißseespitze was also surveyed during federal mapping campaigns and is named and indicated in maps from the third survey of Tyrol onwards, which took place during 1870–1887 and quotes elevation for the rock outcrops and two edges leading down from the summit. Although early measurements of elevation have been challenging and generally have uncertainties of several metres, the data fits remarkably well to the historical photographs and today’s high-resolution data at rock outcrops. Reprocessing the historical map of Hochjochferner based on the original photogrammetric records returned a standard error in elevation of the primary observatory network (triangulation points) of ± 0.21 m40 and the Jungfraujoch temperature records59 , homogenized monthly mean temperatures are available from 1933 onwards. During summer months June, July and August in the overlapping period 1933 to 2014, the difference between Jungfraujoch and Weißseespitze temperature means is 2.3 °C, with a standard deviation of 0.2 °C. This difference was added to the HISTALP monthly mean for a first rough estimate of melting conditions at the summit during the HISTALP period. For the summer means , when most of the melt events occur, the Pearson correlation coefficient between Jungfraujoch and the extrapolated HISTALP grid point is 0.94, for annual means 0.97. As the resulting extrapolated records are very similar Table , S3, we Potential snow accumulation was calculated for the HISTALP grid point from the fraction of solid precipitation and total precipitation, assuming that precipitation in December, January and February was eroded by winds as is the case today. No correction to the altitude was applied to the amount of precipitation or the fraction of solid precipitation.Supplementary Information.


Mortality in severe COVID-19 pneumonia is associated with thrombo-inflammation. Corticosteroids are given to attenuate the inflammation, but they are associated with thrombosis. The aims of this study were to determine the risk of venous thromboembolism between no methylprednisolone and methylprednisolone (dose versus duration) and to evaluate any synergistic dose-dependent association of heparin and methylprednisolone to 30 days in hospital survival.This was a secondary analysis of a retrospective cohort. Patients included in this study were ≥ 18 years of age and admitted for severe COVID-19 pneumonia between March and June 2020 in 13 hospitals in New Jersey, United States. A propensity score analysis between administration of methylprednisolone and no methylprednisolone was fitted for 11 variables and Youden Index Method was used to determine cut-off between low dose and high dose methylprednisolone. Multivariate cox regression was to assess risk.In 759 patients, the incidence of venous thromboembolism was 9% of patients who received methylprednisolone and 3% of patients who did not receive methylprednisolone with a [RR 2.92 ]. There was a higher incidence of mechanical ventilation in the methylprednisolone group. The median d-dimer between patients with venous thromboembolism was higher compared to those without (P < 0.0003). However, the d-dimer was not statistically significant between those who had venous thromboembolism between methylprednisolone and no methylprednisolone groups (P = 0.40). There was no higher risk in high dose versus low dose [RR = 0.524 ]; however, the risk for venous thromboembolism between methylprednisolone for > 7 days and ≤ 7 days was statistically significant . Patients who received low dose methylprednisolone and therapeutic heparin had a trend towards higher risk of mortality compared to prophylactic heparin (HR 1.81 95% CI 0.994 to 3.294) (P = 0.0522). There was no difference in 30 days in hospital survival between high dose methylprednisolone with prophylactic or therapeutic heparin (HR 0.827 95% CI 0.514 to 1.33) (P = 0.4335).Methylprednisolone for > 7 days had a higher association of venous thromboembolism. There was no added benefit of therapeutic heparin to methylprednisolone on mechanically ventilated patients.The online version contains supplementary material available at 10.1186/s12890-021-01810-1. As of July 2, 2021, there has been 182,319,261 coronavirus disease 2019 (COVID-19) cases and 3,954,324 deaths worldwide , atrial fibrillation, or acute coronary syndrome. Therefore, there was a higher proportion of patients on oxygen support who also received therapeutic heparin.9% of patients who received methylprednisolone [LDMP N = 25) and HDMP (N = 10)] and 3% of patients who did not receive methylprednisolone (N = 12), had VTE [RR 2.92 ]. The relative risk between HDMP vs LDMP [RR 0.524 ] was not statistically significant  with no oxygen support, 11 patients (10.19%) with non-invasive oxygen support, and 17 patients (89.47%) with invasive oxygen support or mechanical ventilation expired (P < 0.0001). In patients who received methylprednisolone and prophylactic heparin, 10 patients (16.67%) with no oxygen support, 6 patients (8.70%) with non-invasive oxygen support, and 30 patients 61.22%) with invasive oxygen support expired (P < 0.0001). In patients who received methylprednisolone and therapeutic heparin, 6 patients (25%) with no oxygen support, 9 patients (45%) with non-invasive oxygen support, and 11 patients (78.57%) with invasive oxygen support or mechanical ventilation expired (P = 0.0059) (Additional file 2% with iThere was a higher risk of venous thromboembolism in patients who received methylprednisolone compared to no methylprednisolone use. This may be due to higher production of procoagulant factors from corticosteroids. Cushing’s syndrome, which is due to excess levels of endogenous glucocorticoids, is associated with elevated levels of procoagulant Factor VIII, Factor IX and von Willebrand Factor –35. In vAnother possible explanation is that the association of methylprednisolone with VTE maybe due to the severity of underlying inflammatory disease. Severity may be suggested with higher median CRP and higher oxygen support in the methylprednisolone group compared to no methylprednisolone group. There was a higher frequency of corticosteroids in those requiring invasive mechanical ventilation. This was like the finding from another study from that time, although they used dexamethasone . RegardlOne of the strengths of this study is that we were able to determine which methylprednisolone variable was associated with VTE. HD MP was not associated with higher risk of VTE compared to LD MP, but methylprednisolone > 7 days had a higher risk of VTE compared to ≤ 7 days. This association with VTE may diminish with increasing methylprednisolone use , 21. At Our study suggested therapeutic heparin was not superior to prophylactic heparin, regardless of the methylprednisolone dose to 30 days in hospital survival. Most of these patients required higher level of oxygen support. Our findings are like the conclusions of the two large, randomized trials in non-critically ill and critically ill patients , 30. BotOur study has several limitations. First, since it is an observational study, we cannot draw causal inferences due to known and unknown confounders. However, propensity matching was employed to limit the known confounders. Second, misclassification of data is possible due to manual extraction of structured and unstructured data from medical health records. Third, there was also a higher prevalence of invasive mechanical ventilation, and benefits of therapeutic heparin with methylprednisolone for patients on nasal cannula could not be seen.Duration > 7 days of methylprednisolone is associated with higher risk of venous thromboembolism. This may be due to prolonged duration and severity of inflammation of the underlying disease. Regardless of whether the patient received methylprednisolone or not, higher levels of d-dimer were associated with venous thrombosis. There was no synergistic benefit of methylprednisolone and therapeutic heparin for severe COVID-19 requiring higher oxygen support. Therapeutic heparin should be reserved for patients with radiographic evidence of venous thrombo-embolic disease.Additional file 1. Supplemental Section with Figure S1 and Tables S1–S6.