Identification of genetic variants of the industrial yeast Komagataella phaffii (Pichia pastoris) that contribute to increased yields of secreted heterologous proteins.

The yeast Komagataella phaffii (formerly called Pichia pastoris) is used widely as a host for secretion of heterologous proteins, but only a few isolates of this species exist and all the commonly used expression systems are derived from a single genetic background, CBS7435 (NRRL Y-11430). We hypothesized that other genetic backgrounds could harbor variants that affect yields of secreted proteins. We crossed CBS7435 with 2 other K. phaffii isolates and mapped quantitative trait loci (QTLs) for secretion of a heterologous protein, ß-glucosidase, by sequencing individual segregant genomes. A major QTL mapped to a frameshift mutation in the mannosyltransferase gene HOC1, which gives CBS7435 a weaker cell wall and higher protein secretion than the other isolates. Inactivation of HOC1 in the other isolates doubled ß-glucosidase secretion. A second QTL mapped to an amino acid substitution in IRA1 that tripled ß-glucosidase secretion in 1-week batch cultures but reduced cell viability, and its effects are specific to this heterologous protein. Our results demonstrate that QTL analysis is a powerful method for dissecting the basis of biotechnological traits in nonconventional yeasts, and a route to improving their industrial performance.

A widespread inversion polymorphism conserved among Saccharomyces species is caused by recurrent homogenization of a sporulation gene family.

Saccharomyces genomes are highly collinear and show relatively little structural variation, both within and between species of this yeast genus. We investigated the only common inversion polymorphism known in S. cerevisiae, which affects a 24-kb 'flip/flop' region containing 15 genes near the centromere of chromosome XIV. The region exists in two orientations, called reference (REF) and inverted (INV). Meiotic recombination in this region is suppressed in crosses between REF and INV orientation strains such as the BY x RM cross. We find that the inversion polymorphism is at least 17 million years old because it is conserved across the genus Saccharomyces. However, the REF and INV isomers are not ancient alleles but are continually being re-created by re-inversion of the region within each species. Inversion occurs due to continual homogenization of two almost identical 4-kb sequences that form an inverted repeat (IR) at the ends of the flip/flop region. The IR consists of two pairs of genes that are specifically and strongly expressed during the late stages of sporulation. We show that one of these gene pairs, YNL018C/YNL034W, codes for a protein that is essential for spore formation. YNL018C and YNL034W are the founder members of a gene family, Centroid, whose members in other Saccharomycetaceae species evolve fast, duplicate frequently, and are preferentially located close to centromeres. We tested the hypothesis that Centroid genes are a meiotic drive system, but found no support for this idea.

Fully integrated downstream process to enable next-generation manufacturing.

Next-generation manufacturing (NGM) has evolved over the past decade to a point where large biopharmaceutical organizations are making large investments in the technology and considering implementation in clinical and commercial processes. There are many well-considered reasons to implement NGM. For the most part, organizations will not fund NGM unless the implementation benefits the funding organization by providing reduced costs, reduced time, or additional needed capabilities. Productivity improvements gained from continuous purification are shown in this work, which used a new system that fully integrates and automates several downstream unit operations of a biopharmaceutical process to provide flexibility and easy implementation of NGM. The equipment and automation needed to support NGM can be complicated and expensive. Biopharmaceutical Process Development considered two options as follows: (1) design its own NGM system or (2) buy a prebuilt system. PAK BioSolutions offers a turn-key automated and integrated system that can operate up to four continuous purification stages simultaneously, while maintaining a small footprint in the manufacturing plant. The system provides significant cost benefits (~10× lower) compared with the alternative-integration of many different pieces of equipment through a Distributed Control System that would require significant engineering time for design, automation, and integration. Integrated and Continuous Biomanufacturing can lead to significant reductions in facility size, reduced manufacturing costs, and enhanced product quality when compared with the traditional batch mode of operation. The system uses new automation strategies that robustly link unit operations. We present the optimized process fit, sterility and bioburden control strategy, and automation features (such as pH feedback control and in-line detergent addition), which enabled continuous operation of a 14-day end-to-end monoclonal antibody purification process at the clinical manufacturing scale.

Posterior cruciate ligament rupture and all-epiphyseal repair with suture tape augmentation in a 5-year-old girl: a case report and review of the literature.

INTRODUCTION: Only a few case reports regarding pediatric posterior cruciate ligament (PCL) ruptures without bone avulsion exist in the literature. The present study aims to share our experience in the diagnosis, treatment, and prognosis of a child with a proximal PCL tear. MATERIALS AND METHODS: This article reports a 5-year-old female diagnosed with a proximal PCL tear. The ruptured PCL was repaired with an all-epiphyseal suture tape augmentation (STA) without evidence of growth plate violation. RESULTS: The suture tape was removed under arthroscopy and revealed the PCL was re-attached at 12 months after the first surgery. And at the time of this report, 36 months after surgery, she was doing well without any problems and with negative posterior drawer test. CONCLUSIONS: Pediatric PCL tear without bone avulsion is rare. However, the torn PCL was noticed healed based on an arthroscopic second-look.

Identification of European isolates of the lager yeast parent Saccharomyces eubayanus.

Lager brewing first occurred in Bavaria in the 15th century, associated with restrictions of brewing to colder months. The lager yeast, Saccharomyces pastorianus, is cold tolerant. It is a hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus, and has been found only in industrial settings. Natural isolates of S. eubayanus were first discovered in Patagonia 11 years ago. They have since been isolated from China, Tibet, New Zealand, and North America, but not from Europe. Here, we describe the first European strains UCD646 and UCD650, isolated from a wooded area on a university campus in Dublin, Ireland. We generated complete chromosome level assemblies of both genomes using long- and short-read sequencing. The UCD isolates belong to the Holarctic clade. Genome analysis shows that isolates similar to the Irish strains contributed to the S. eubayanus component of S. pastorianus, but isolates from Tibet made a larger contribution.

Provocative tests with Kisspeptin-10 and GnRH set the scene for determining social status and environmental impacts on reproductive capacity in male African lions (Panthera leo).

Understanding the hypothalamic factors regulating reproduction facilitates maximising the reproductive success of breeding programmes and in the management and conservation of threatened species, including African lions. To provide insight into the physiology and pathophysiology of the hypothalamic-pituitary-gonadal reproductive axis in lions, we studied the luteinising hormone (LH) and steroid hormone responses to gonadotropin-releasing hormone (GnRH) and its upstream regulator, kisspeptin. Six young (13.3 ± 1.7 months, 56.2 ± 4.3 kg) and four adult (40.2 ± 1.4 months, 174 ± 6 kg) male lions (Ukutula Conservation Centre, South Africa) were used in this study. Lions were immobilised with a combination of medetomidine and ketamine and an intravenous catheter was placed in a jugular, cephalic or medial saphenous vein for blood sampling at 10-min intervals for 220 min. The ten-amino acid kisspeptin which has full intrinsic activity (KP-10, 1 µg/kg) and GnRH (1 µg/kg) were administered intravenously to study their effects on LH and steroid hormone plasma concentrations, measured subsequently by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS), respectively. Basal LH levels were similarly low between the age groups, but testosterone and its precursor levels were higher in the adult animals. Adult lions showed a significant LH response to KP-10 (10-fold) and GnRH (11-fold) administration (p < 0.05 and P < 0.001, respectively) whereas in young lions LH increased significantly only in response to GnRH. In adults alone, testosterone and its precursors steadily increased in response to KP-10, with no significant further increase in response to GnRH. Plasma levels of glucocorticoids in response to KP-10 remained unchanged. We suggest that provocative testing of LH and steroid stimulation with kisspeptin provides a new and sensitive tool for determining reproductive status and possibly an index of exposure to stress, environmental insults such as disease, endocrine disruptors and nutritional status. 272 words.

Two-fragment Segond fracture validates historical descriptions of independent soft tissue attachments.

This is a case report of a 26-year-old male who sustained a Segond fracture in the context of an acute anterior cruciate ligament (ACL) rupture incurred while downhill skiing. Further work-up revealed that the Segond fracture consisted of two distinct fragments with separate soft tissue attachments, including the capsule-osseous layer of the iliotibial band and the short arm of the biceps femoris. Imaging showed interval healing of the Segond fracture between initial presentation and the performance of arthroscopic ACL reconstruction approximately 4 months later. As intraoperative evaluation demonstrated that anatomic ACL reconstruction restored translational and rotatory knee stability, surgical repair of the Segond fracture, or the anterolateral complex of the knee more broadly, was not required. Maintenance of translational and rotatory knee stability was confirmed at serial post-operative appointments up through final follow-up.Level of evidence Level V.

Knees with straight Blumensaat's line have small volume of femoral intercondylar notch.

PURPOSE: Smaller femoral intercondylar notch volume has been identified as a risk factor for anterior cruciate ligament injury. The present study aims to investigate differences in the intercondylar notch volume based on differences in the morphology of Blumensaat's line. METHODS: Eighty-eight (88) subjects (42 male and 46 female: median age 27: range 15-49), were included in this study. Using 3-dimensional computed tomography (3D-CT), the volume of the intercondylar notch was calculated using a truncated-pyramid shape simulation with the formula: [Formula: see text]. Femoral condyle height (h) was measured in the sagittal plane of the knee in 3D-CT. The area of the intercondylar notch was measured in the axial slice containing the most proximal level (S1) and most distal level (S2) of Blumensaat's line. In the sagittal view of the knee, Blumensaat's line morphology was classified into either straight or hill type. Statistical analysis was performed to compare h, S1, S2, and notch volume between the straight and hill type groups. RESULTS: Thirty-six subjects were classified as having straight type morphology and 52 subjects were classified as having hill type morphology. The measured h, S1, and S2, of the straight and hill types were 29 ± 4 and 31 ± 4 mm, 213 ± 72 and 205 ± 51 mm2, 375 ± 114 and 430 ± 94 mm2, respectively. The calculated femoral intercondylar notch volume of the straight and hill types was 8.1 ± 2 and 9.5 ± 2 cm3, respectively. Straight type knees showed significantly smaller S2 (p = 0.04), and notch volume (p = 0.01) when compared with hill type knees. CONCLUSION: Intercondylar notch volume was significantly smaller in knees with straight type Blumensaat's line morphology. Considering that Blumensaat's line represents the roof of the femoral notch, morphological variations in Blumensaat's line are likely to reflect variation in notch volume. For clinical relevance, as a smaller notch volume is a risk factor for ACL injury, straight type Blumensaat's line may also be considered a potential risk factor for ACL injury. LEVEL OF EVIDENCE: Level III.

Low to moderate risk of nerve damage during peroneus longus tendon autograft harvest.

PURPOSE: This study aims to evaluate the proximity of the tendon stripper to both the peroneal and sural nerves during peroneus longus tendon (PLT) autograft harvesting. METHODS: Ten fresh-frozen human cadaveric lower extremities were used to harvest a full-thickness PLT autograft using a standard closed blunt-ended tendon stripper. The distance to the sural nerve from the PLT (at 0, 1, 2 and 3 cm proximal to lateral malleolus (LM), and the distance to the peroneal nerve and its branches from the end of the tendon stripper were measured by two separate observers using ImageJ software. RESULTS: The average distance from the PLT to the sural nerve increased significantly from 0 to 2 cm proximal to LM. The average distance to the sural nerve at the LM was 4.9 ± 1.5 mm and increased to 10.8 ± 2.4 mm (2 cm proximal to LM). The average distance from the tendon stripper to the deep peroneal nerve was 52.9 ± 11.4 mm. The average distance to the PLT branch of peroneal nerve was 29.3 ± 4.2 mm. The superficial peroneal nerve, which coursed parallel and deep to the tendon stripper, was on average 5.2 ± 0.7 mm from the end of the stripper. No transection injuries of the nerves were observed in any of the ten legs after harvesting. CONCLUSION: This cadaver study found during a full-thickness PLT harvest, the distances between the tendon stripper and the nerves were greater than 5 mm with an initial incision at 2 cm proximal to LM which is recommended.

Non-anatomic tunnel position increases the risk of revision anterior cruciate ligament reconstruction.

PURPOSE: Anterior cruciate ligament (ACL) graft failure is a complication that may require revision ACL reconstruction (ACL-R). Non-anatomic placement of the femoral tunnel is thought to be a frequent cause of graft failure; however, there is a lack of evidence to support this belief. The purpose of this study was to determine if non-anatomic femoral tunnel placement is associated with increased risk of revision ACL-R. METHODS: After screening all 315 consecutive patients who underwent primary single-bundle ACL-R by a single senior orthopedic surgeon between January 2012 and January 2017, 58 patients were found to have both strict lateral radiographs and a minimum of 24 months follow-up without revision. From a group of 456 consecutive revision ACL-R, patients were screened for strictly lateral radiographs and 59 patients were included in the revision group. Femoral tunnel placement for each patient was determined using a strict lateral radiograph taken after the primary ACL-R using the quadrant method. The center of the femoral tunnel was measured in both the posterior-anterior (PA) and proximal-distal (PD) dimensions and represented as a percentage of the total distance (normal center of anatomic footprint: PA 25% and PD 29%). RESULTS: In the PA dimension, the revision group had significantly more anterior femoral tunnel placement compared with the primary group (38% ± 11% vs. 28% ± 6%, p < 0.01). Among patients who underwent revision; those with non-traumatic chronic failure had statistically significant more anterior femoral tunnel placement than those who experienced traumatic failure (41% ± 13% vs. 35% ± 8%, p < 0.03). In the PD dimension, the revision group had significantly more proximal femoral tunnel placement compared with the primary group (30% ± 9% vs 38% ± 9%, p < 0.01). CONCLUSION: In this retrospective study of 58 patients with successful primary ACL-R compared with 59 patients with failed ACL-R, anterior and proximal (high) femoral tunnels for ACL-R were shown to be independent risk factors for ACL revision surgery. As revision ACL-R is associated with patient- and economic burden, particular attention should be given to achieving an individualized, anatomic primary ACL-R. Surgeons may reduce the risk of revision ACL-R by placing the center of the femoral tunnel within the anatomic ACL footprint. LEVEL OF EVIDENCE: Level III.

Editorial Commentary: Outcomes After Anterior Cruciate Ligament Reconstruction Are Defined by Individual Anatomy, Including Both Soft Tissue and Bone Morphology: It's All Important.

Well-designed studies add to our understanding of the anatomy, biology, biomechanics, and outcomes of the anterior cruciate ligament (ACL) following injury. Despite improvements in ACL treatment, we are still unable to exactly restore the individually unique function of the native ACL due to the complexity of knee physiology. The ACL is a dynamic structure with a rich neurovascular supply, distinct bundles, and 3-dimensional architecture that function in synergy with the bony morphology to facilitate healthy knee kinematics. Furthermore, the ACL exhibits a wide range of natural, anatomic variation. Since anatomic ACL reconstruction has been defined as functional restoration of the ACL to its native dimensions and collagen orientation, in addition to restoring the native footprint, it is important to restore the native size of the ACL, as the size of the tibial insertion site can vary by a factor of 3 from patient to patient. Moreover, variations in ACL soft tissue reflect differences in bony morphology. Bony morphology influences the static and dynamic biomechanics of the knee. Several bony morphologic factors influence the outcomes following ACL reconstruction, including posterior tibial slope, femoral condylar offset ratio, and notch shape. Morphologic differences that reflect pathologic states, such as the lateral notch sign and posterolateral plateau fracture, have been shown to be associated with greater grade instability. To respect the unique nature of each patient during surgical treatment, it is necessary to perform an individualized, anatomic, and value-based ACL reconstruction.

Editorial Commentary: Remember the Risk Factors During Individualized, Anatomic, Value-Based Anterior Cruciate Ligament Reconstruction.

Understanding the etiology behind anterior cruciate ligament (ACL) reconstruction failure is a complex topic still being investigated heavily. The 3 classes of failure are technical, traumatic, and biologic. Technical errors are most common and most frequently reflect tunnel malposition. In addition, tibial slope has long been understood to be a risk factor for failed ACL reconstruction. Although not routinely performed at time of primary ACL reconstruction, osteotomy may be considered in the setting of failed ACL reconstruction. Relative quadriceps weakness is a risk factor, and we recommend sport-specific return-to-play testing as well as benchmarks for relative quadriceps strength before full return to activity. Revision ACL reconstruction is associated with both increased costs and worse patient outcomes, so every effort should be made to give patients the best chance of success after the index surgery. Whereas this begins with understanding the patient's history and risk factors for failure, it crescendos with careful attention to the individually variable factors that make each case unique, tailoring one's management to ensure that each patient receives an anatomic, individualized, and value-based ACL reconstruction.

The Origin of GnRH Pulse Generation: An Integrative Mathematical-Experimental Approach.

Fertility critically depends on the gonadotropin-releasing hormone (GnRH) pulse generator, a neural construct comprised of hypothalamic neurons coexpressing kisspeptin, neurokoinin-B and dynorphin. Here, using mathematical modeling and in vivo optogenetics we reveal for the first time how this neural construct initiates and sustains the appropriate ultradian frequency essential for reproduction. Prompted by mathematical modeling, we show experimentally using female estrous mice that robust pulsatile release of luteinizing hormone, a proxy for GnRH, emerges abruptly as we increase the basal activity of the neuronal network using continuous low-frequency optogenetic stimulation. Further increase in basal activity markedly increases pulse frequency and eventually leads to pulse termination. Additional model predictions that pulsatile dynamics emerge from nonlinear positive and negative feedback interactions mediated through neurokinin-B and dynorphin signaling respectively are confirmed neuropharmacologically. Our results shed light on the long-elusive GnRH pulse generator offering new horizons for reproductive health and wellbeing.SIGNIFICANCE STATEMENT The gonadotropin-releasing hormone (GnRH) pulse generator controls the pulsatile secretion of the gonadotropic hormones LH and FSH and is critical for fertility. The hypothalamic arcuate kisspeptin neurons are thought to represent the GnRH pulse generator, since their oscillatory activity is coincident with LH pulses in the blood; a proxy for GnRH pulses. However, the mechanisms underlying GnRH pulse generation remain elusive. We developed a mathematical model of the kisspeptin neuronal network and confirmed its predictions experimentally, showing how LH secretion is frequency-modulated as we increase the basal activity of the arcuate kisspeptin neurons in vivo using continuous optogenetic stimulation. Our model provides a quantitative framework for understanding the reproductive neuroendocrine system and opens new horizons for fertility regulation.

Population genomics shows no distinction between pathogenic Candida krusei and environmental Pichia kudriavzevii: One species, four names.

We investigated genomic diversity of a yeast species that is both an opportunistic pathogen and an important industrial yeast. Under the name Candida krusei, it is responsible for about 2% of yeast infections caused by Candida species in humans. Bloodstream infections with C. krusei are problematic because most isolates are fluconazole-resistant. Under the names Pichia kudriavzevii, Issatchenkia orientalis and Candida glycerinogenes, the same yeast, including genetically modified strains, is used for industrial-scale production of glycerol and succinate. It is also used to make some fermented foods. Here, we sequenced the type strains of C. krusei (CBS573T) and P. kudriavzevii (CBS5147T), as well as 30 other clinical and environmental isolates. Our results show conclusively that they are the same species, with collinear genomes 99.6% identical in DNA sequence. Phylogenetic analysis of SNPs does not segregate clinical and environmental isolates into separate clades, suggesting that C. krusei infections are frequently acquired from the environment. Reduced resistance of strains to fluconazole correlates with the presence of one gene instead of two at the ABC11-ABC1 tandem locus. Most isolates are diploid, but one-quarter are triploid. Loss of heterozygosity is common, including at the mating-type locus. Our PacBio/Illumina assembly of the 10.8 Mb CBS573T genome is resolved into 5 complete chromosomes, and was annotated using RNAseq support. Each of the 5 centromeres is a 35 kb gene desert containing a large inverted repeat. This species is a member of the genus Pichia and family Pichiaceae (the methylotrophic yeasts clade), and so is only distantly related to other pathogenic Candida species.

Evolutionary restoration of fertility in an interspecies hybrid yeast, by whole-genome duplication after a failed mating-type switch.

Many interspecies hybrids have been discovered in yeasts, but most of these hybrids are asexual and can replicate only mitotically. Whole-genome duplication has been proposed as a mechanism by which interspecies hybrids can regain fertility, restoring their ability to perform meiosis and sporulate. Here, we show that this process occurred naturally during the evolution of Zygosaccharomyces parabailii, an interspecies hybrid that was formed by mating between 2 parents that differed by 7% in genome sequence and by many interchromosomal rearrangements. Surprisingly, Z. parabailii has a full sexual cycle and is genetically haploid. It goes through mating-type switching and autodiploidization, followed by immediate sporulation. We identified the key evolutionary event that enabled Z. parabailii to regain fertility, which was breakage of 1 of the 2 homeologous copies of the mating-type (MAT) locus in the hybrid, resulting in a chromosomal rearrangement and irreparable damage to 1 MAT locus. This rearrangement was caused by HO endonuclease, which normally functions in mating-type switching. With 1 copy of MAT inactivated, the interspecies hybrid now behaves as a haploid. Our results provide the first demonstration that MAT locus damage is a naturally occurring evolutionary mechanism for whole-genome duplication and restoration of fertility to interspecies hybrids. The events that occurred in Z. parabailii strongly resemble those postulated to have caused ancient whole-genome duplication in an ancestor of Saccharomyces cerevisiae.

Fluorescent supramolecular hierarchical self-assemblies from glycosylated 4-amino- and 4-bromo-1,8-naphthalimides.

An investigation into the self-assembly of two 4-amino- and a 4-bromo-1,8-naphthalimide (Nap) based structures (1-3) possessing an appended glycan unit, from protic polar media, is presented. The results demonstrate the formation of complex hierarchical luminescent aggregates, wherein the morphologies, sizes and spherical structures were highly dependent on both the media and the Nap structure. Upon cleaving the native glycosidic bond, using an enzyme, the structure/morphology of the self-assembly of 3 in buffered solution was significantly transformed.

Flip/flop mating-type switching in the methylotrophic yeast Ogataea polymorpha is regulated by an Efg1-Rme1-Ste12 pathway.

In haploid cells of Ogataea (Hansenula) polymorpha an environmental signal, nitrogen starvation, induces a reversible change in the structure of a chromosome. This process, mating-type switching, inverts a 19-kb DNA region to place either MATa or MATα genes under centromeric repression of transcription, depending on the orientation of the region. Here, we investigated the genetic pathway that controls switching. We characterized the transcriptomes of haploid and diploid O. polymorpha by RNAseq in rich and nitrogen-deficient media, and found that there are no constitutively a-specific or α-specific genes other than the MAT genes themselves. We mapped a switching defect in a sibling species (O. parapolymorpha strain DL-1) by interspecies bulk segregant analysis to a frameshift in the transcription factor EFG1, which in Candida albicans regulates filamentous growth and white-opaque switching. Gene knockout, overexpression and ChIPseq experiments show that EFG1 regulates RME1, which in turn regulates STE12, to achieve mating-type switching. All three genes are necessary both for switching and for mating. Overexpression of RME1 or STE12 is sufficient to induce switching without a nitrogen depletion signal. The homologous recombination genes RAD51 and RAD17 are also necessary for switching. The pathway controlling switching in O. polymorpha shares no components with the regulation of HO in S. cerevisiae, which does not involve any environmental signal, but it shares some components with mating-type switching in Kluyveromyces lactis and with white-opaque phenotypic switching in C. albicans.

Effects of focal metallic implants on opposing cartilage - an in-vitro study with an abrasion test machine.

BACKGROUND: For focal cartilage defects, biological repair might be ineffective in patients over 45 years. A focal metallic implant (FMI) (Hemi-CAP Arthrosurface Inc., Franklin, MA, USA) was designed to reduce symptoms. The aim of this study was to evaluate the effects of a FMI on the opposing tibial cartilage in a biomechanical set-up. It is hypothesized that a FMI would not damage the opposing cartilage under physiological loading conditions. METHODS: An abrasion machine was used to test the effects of cyclic loading on osteochondral plugs. The machine applied a compressive load of 33 N and sheared the samples 10 mm in the anteroposterior direction by 1 Hz. Tibial osteochondral plugs from porcine knees were placed in opposition to a FMI and cycled for 1 or 6 h. After testing each plug was fixed, stained and evaluated for cartilage damage. RESULTS: After 1 h of loading (n = 6), none of the osteochondral plugs showed histologic signs of degradation. After 6 h of loading (n = 6) three samples had histologic signs of injury in the tangential zone (grade 1) and one had signs of injury in the transitional and deep zones (grade 2). Exploration for 6 h resulted in significant more cartilage damage compared to the shorter exploration time (p = 0.06). However, no significant difference between saline and hyaluronic acid was evident (p = 0.55). CONCLUSION: Under physiologic loading conditions, contact with a FMI leads to cartilage damage in the opposing articular cartilage in six hours. In clinical practice, a thorough analysis of pre-existing defects on the opposing cartilage is recommended when FMI is considered.

The Methylotroph Gene Order Browser (MGOB) reveals conserved synteny and ancestral centromere locations in the yeast family Pichiaceae.

The yeast family Pichiaceae, also known as the 'methylotrophs clade', is a relatively little studied group of yeasts despite its economic and clinical relevance. To explore the genome evolution and synteny relationships within this family, we developed the Methylotroph Gene Order Browser (MGOB, http://mgob.ucd.ie) similar to our previous gene order browsers for other yeast families. The dataset contains genome sequences from nine Pichiaceae species, including our recent reference sequence of Pichia kudriavzevii. As an example, we demonstrate the conservation of synteny around the MOX1 locus among species both containing and lacking the MOX1 gene for methanol assimilation. We found ancient clusters of genes that are conserved as adjacent between Pichiaceae and Saccharomycetaceae. Surprisingly, we found evidence that the locations of some centromeres have been conserved among Pichiaceae species, and between Pichiaceae and Saccharomycetaceae, even though the centromeres fall into different structural categories-point centromeres, inverted repeats and retrotransposon cluster centromeres.

Genomic diversity and meiotic recombination among isolates of the biotech yeast Komagataella phaffii (Pichia pastoris).

BACKGROUND: Komagataella phaffii is a yeast widely used in the pharmaceutical and biotechnology industries, and is one of the two species that were previously called Pichia pastoris. However, almost all laboratory work on K. phaffii has utilized strains derived from a single natural isolate, CBS7435. There is little information about the sequence diversity of K. phaffii or the genetic properties of this species. RESULTS: We sequenced the genomes of all the known isolates of K. phaffii. We made a genetic cross between derivatives of two isolates that differ at 44,000 single nucleotide polymorphism sites, and used this cross to analyze the rate and landscape of meiotic recombination. We conducted tetrad analysis by making use of the property that K. phaffii haploids do not mate in rich media, which enabled us to isolate and sequence the four types of haploid cell that are present in the colony that forms when a tetra-type ascus germinates. CONCLUSIONS: We found that only four distinct natural isolates of K. phaffii exist in public yeast culture collections. The meiotic recombination rate in K. phaffii is approximately 3.5 times lower than in Saccharomyces cerevisiae, with an average of 25 crossovers per meiosis. Recombination is suppressed, and genetic diversity among natural isolates is low, in a region around centromeres that is much larger than the centromeres themselves. Our work lays a foundation for future quantitative trait locus analysis in K. phaffii.