From the Cover: 1.36 million years of Mediterranean forest refugium dynamics in response to glacial–interglacial cycle strength

The sediment record from Lake Ohrid (Southwestern Balkans) represents the longest continuous lake archive in Europe, extending back to 1.36 Ma. We reconstruct the vegetation history based on pollen analysis of the DEEP core to reveal changes in vegetation cover and forest diversity during glacial–interglacial (G–IG) cycles and early basin development. The earliest lake phase saw a significantly different composition rich in relict tree taxa and few herbs. Subsequent establishment of a permanent steppic herb association around 1.2 Ma implies a threshold response to changes in moisture availability and temperature and gradual adjustment of the basin morphology. A change in the character of G–IG cycles during the Early–Middle Pleistocene Transition is reflected in the record by reorganization of the vegetation from obliquity- to eccentricity-paced cycles. Based on a quantitative analysis of tree taxa richness, the first large-scale decline in tree diversity occurred around 0.94 Ma. Subsequent variations in tree richness were largely driven by the amplitude and duration of G–IG cycles. Significant tree richness declines occurred in periods with abundant dry herb associations, pointing to aridity affecting tree population survival. Assessment of long-term legacy effects between global climate and regional vegetation change reveals a significant influence of cool interglacial conditions on subsequent glacial vegetation composition and diversity. This effect is contrary to observations at high latitudes, where glacial intensity is known to control subsequent interglacial vegetation, and the evidence demonstrates that the Lake Ohrid catchment functioned as a refugium for both thermophilous and temperate tree species.

Identification and protection of past forest refugia, supporting a relict population, has gained interest in light of projected forest responses to anthropogenic climate change (1–4). Understanding the past and present composition of Mediterranean forest refugia is central to the study of long-term survival of tree taxa and the systematic relation between forest dynamics and climate (5). The Quaternary vegetation history of Europe, studied for over a century, is characterized by successive loss of tree species (6–8). Species loss was originally explained by the repeated migration across east–west oriented mountain chains during glacial–interglacial (G–IG) cycles (9). Later views gave more importance to the survival of tree populations during warm and arid stages in southern refugia (10, 11). Tree survival likely depends on persistence of suitable climate and tolerable levels of climate variability, as well as niche differentiation and population size at the refugium (12), although the precise relation between regional extinctions, climate variability, and local edaphic factors is not well known (13). Mediterranean mountain regions are considered to serve as forest refugia over multiple glacial cycles and frequently coincide with present-day biodiversity hotspots (14). Across the Mediterranean, increases in aridity and fire occurrence have impacted past vegetation communities (15–18). Comprehensive review of available Quaternary Mediterranean records indicates that Early (2.58 to 0.77 Ma) and Middle Pleistocene (0.77 to 0.129 Ma) tree diversity was higher compared to the present (13, 19–21). Particularly drought intolerant, thermophilic taxa were more abundant and diverse (8) but with strong spatial and temporal variations in tree diversity across the region. Long-term relationships between refugia function and environmental change over multiple G–IG cycles are hard to quantify due to the rarity of long, uninterrupted records.The Early–Middle Pleistocene Transition (EMPT), between 1.4 and 0.4 Ma (22), is of particular importance for understanding the relation between past climate change, vegetation dynamics, and biodiversity in the Mediterranean region. The EMPT is characterized by a gradual transition of G–IG cycle duration from obliquity (41 thousand years; kyr) to eccentricity (100 kyr) scale with increasing amplitude of each G–IG cycle (e.g., refs. 23, 24). The EMPT was accompanied by long-term cooling of the deep and surface ocean and was likely caused by atmospheric CO₂ decline and ice-sheet feedbacks (25–30). In Europe, the EMPT is associated with pronounced vegetation changes and local extinction and isolation of small tree populations (31).Here, we document vegetation history of the last 1.36 Ma in the Lake Ohrid (LO) catchment, located at the Albanian/North Macedonian border at 693 m above sea level (m asl, Fig. 1), the longest continuous sedimentary lake record in Europe (32, 33). The chronology of the DEEP core (International Continental Scientific Drilling Program site 5045-1; 41°02’57’’ N, 20°42’54’’ E, Fig. 1) is based on tuning of biogeochemical proxy data to orbital parameters with independent tephrostratigraphic and paleomagnetic age control (32, 33). The Balkan Peninsula has long been considered an important glacial forest refugium for presently widespread taxa such as Abies, Picea, Carpinus, Corylus, Fagus Ostrya, Quercus, Tilia, and Ulmus (7, 34–36). More than 60% of the Balkans is currently located >1,000 m asl (36), providing steep latitudinal and elevational gradients to support refugia under both cold and warm conditions. Today, the LO catchment is dominated by (semi) deciduous oak (Quercus) and hornbeam (Carpinus/Ostrya) forests. Above 1,250 m elevation, mixed mesophyllous forest with montane elements occurs (Fagus and at higher elevations Abies), which above 1,800 m elevation develops into subalpine grassland with Juniperus shrubs (see ref. 37 for site details). Isolated populations of Pinus peuce and Pinus nigra currently grow in the area (37–40).Open in a separate windowFig. 1.(A) Location of LO and TP on the Balkan Peninsula. (B) Local setting around LO, bathymetry (81), and DEEP coring site (adapted from ref. 32).Previous analysis of pollen composition of the last 500 kyr at the DEEP site revealed that the LO has been an important refugium. Arboreal pollen (AP) is deposited continuously and changes in abundance on multimillennial timescales in association with G–IG cycles, whereas millennial-scale variability is tightly coupled to Mediterranean sea-surface temperature variations (37, 41–45). Subsequent studies confirm the refugial character of the site recording Early Pleistocene (1.365 to 1.165 Ma) high relict tree diversity and abundance—and significant hydrological changes, including an increase in lake size and depth (38). Here, we present a continuous palynological record from LO with millennial resolution (∼2 kyr) back to 1.36 Ma to assess the systematic relationships between tree pollen abundance, forest diversity, and G–IG climate variability.Our objective is as follows: 1) infer the impact of past climate variability on local vegetation across the EMPT, 2) estimate tree species diversity in the catchment, and 3) examine how the amplitude and duration of preceding G–IG intervals affected the vegetation development and plant species diversity in this refugial area.     

The release and induced immune responses of a plant-made and delivered antigen in the mouse gut

This study investigated the site of release of a model vaccine antigen from plant cells and the corresponding induced immune response. Three plant tissues (leaf, fruit and hairy root) and two formulations (aqueous and lipid) were compared in two mouse trials. A developed technique that enabled detection of antigen release by plant cells determined that antigen release occurred at early sites of the gastrointestinal tract when delivered in leaf material and at later sites when delivered in hairy roots. Lipid formulations delayed antigen release from all plant materials tested. While encapsulation in the plant cell provided some protection of the antigen in the gastrointestinal tract and influenced antigen release, formulation medium was also an important consideration with regard to vaccine delivery and immunogenicity. Systemic immune responses induced from the orally delivered vaccine benefited from late release of antigen in the mouse gastrointestinal tract. The influences to the mucosal immune response induced by these vaccines were too complex to be determined by studies performed here with no clear trend regarding plant tissue site of release or formulation medium. Expression and delivery of the model antigen in plant material prepared in an aqueous formulation provided the optimal systemic and mucosal, antigen-specific immune responses.     

Comparison of fixed dose pegfilgrastim and daily filgrastim after autologous stem cell transplantation in patients with multiple myeloma autografted on a outpatient basis

Different authors have explored the feasibility of autografting patients with multiple myeloma (MM) on an outpatient basis. Peg-filgrastim (PEG), a long-acting recombinant G-CSF, has similar efficacy when compared to conventional G-CSF for chemotherapy-induced neutropenia, but little is known about its use in the autologous stem-cell transplantation (ASCT) setting, namely in patients programmed to be autografted on outpatient basis. In this study, we compared therapeutic results in terms of hematopoietic recovery, non-hematologic toxicity, duration of hospitalization and percentage of hospital readmission between patients receiving either conventional G-CSF or PEG. Thirty-eight MM patients (48 autografts) received PEG, given at a single dose of 6 mg at day +5 from stem cell infusion, while 81 (113 autografts) received G-CSF from day + 2 up to stable neutrophil recovery. The conditioning regimen was high dose melphalan in all patients. The median age and the median number of CD34 + cell infused were comparable between the two groups. Overall, a second hospital admission was required in 36 procedures out of 161 (32%). Febrile neutropenia (FN) and severe mucositis were the most frequent causes of hospitalization. There was no statistically significant difference as percentage of hospital readmission is concerned: in the PEG group readmission was needed in 6 out of 48 autografts (12%) as opposed to 30 out of 113 (26%) in the G-CSF subgroup, p: 0.06. The median time of hospital stay for readmitted patients was identical for the two subgroups (9 days vs. 9 days, p: 0.94). Finally, one case of transplant related mortality occurred in the whole patient series (0.6%). In conclusion, ASCT on an outpatient basis is feasible and safe in patients with MM, the majority of whom are manageable at home. The administration of single dose PEG results in no different outcome in terms of safety and efficacy as compared to 8 days of G-CSF.     

Benign granular cell tumor of the vulva: first report of multiple cases in a family

Granular cell tumors (GCTs) are uncommon soft tissue tumors of neural derivation, as supported by immunohistochemical and ultrastructural evidence. Vulvar involvement has been reported in 7-16%. This paper presents the cases of a 60-year-old woman and her 32-year-old niece with a strong family history of cancer, both presenting with an enlarging mass on their left labia majora. The lesions were treated by simple surgical excision. Histopathological examination revealed a benign vulvar GCT in both lesions. This is the first reported case of GCT of the vulva in the same family. The possible familial component of GCT needs further investigation. A systematic review of the literature on vulvar GCTs is carried out, the most complete one to date. This review unexpectedly reveals that there have been more than 130 cases of GCT of the vulva reported to date, only 7 of which were malignant. Since 5-25% of patients have multiple lesions, before planning treatment, clinicians should exclude multicentric lesions. After surgical treatment, if there is any evidence of tumor in the surgical margin, wider local excision should be performed. Regular follow-up is important for diagnosing a possible recurrence or a new lesion.     

Carbon monoxide contributes to the constipating effects of granisetron in rat colon

AIM To investigate the mechanisms underlying the potential contribution of the heme oxygenase/carbon monoxide(HO/CO) pathway in the constipating effects of granisetron. METHODS For in vivo studies, gastrointestinal motility was evaluated in male rats acutely treated with granisetron [25, 50, 75 μg/kg/subcutaneous(sc)], zinc protoporphyrin IX [Zn PPIX, 50 μg/kg/intraperitoneal(ip)] and hemin(50 μmol/L/kg/ip), alone or in combination. For in vitro studies, the contractile neurogenic response to electrical field stimulation(EFS, 3, 5, 10 Hz, 14 V, 1 ms, pulse trains lasting 10 s), as well as the contractile myogenic response to acetylcholine(ACh, 0.1-100 μmol/L) were evaluated on colon specimens incubated with granisetron(3 μmol/L, 15 min), Zn PPIX(10 μmol/L, 60 min) or CO-releasing molecule-3(CORM-3, 100, 200, 400 μmol/L) alone or in combination. These experiments were performed under co-treatment withor without atropine(3 μmol/L, a muscarinic receptor antagonist) or NG-nitro-L-Arginine(L-NNA, 100 μmol/L, a nitric oxide synthase inhibitor).RESULTS Administration of granisetron(50, 75 μg/kg) in vivo significantly increased the time to first defecation(P = 0.045 vs vehicle-treated rats), clearly suggesting a constipating effect of this drug. Although administration of Zn PPIX or hemin alone had no effect on this gastrointestinal motility parameter, Zn PPIX co-administered with granisetron abolished the granisetron-induced constipation. On the other hand, co-administration of hemin and granisetron did not modify the increased constipation observed under granisetron alone. When administered in vitro, granisetron alone(3 μmol/L) did not significantly modify the colon's contractile response to either EFS or ACh. Incubation with Zn PPIX alone(10 μmol/L) significantly reduced the colon's contractile response to EFS(P = 0.016) but had no effect on contractile response to ACh. Co-administration of Zn PPIX and atropine(3 μmol/L) abolished the Zn PPIX-mediated decrease in contractile response to EFS. Conversely, incubation with CORM-3(400 μmol/L) alone increased both the contractile response to EFS at 10 Hz(10 Hz: 71.02 ± 19.16 vs 116.25 ± 53.70, P = 0.01) and the contractile response to ACh(100 μmol/L)(P = 0.012). Co-administration of atropine abolished the CORM-3-mediated effects on the EFS-mediated response. When granisetron was co-incubated in vitro with ZnP PIX, the ZnP PIX-mediated decrease in colon contractile response to EFS was lost. On the other hand, co-incubation of granisetron and CORM-3(400 μmol/L) further increased the colon's contractile response to EFS(at 5 Hz: P = 0.007; at 10 Hz: P = 0.001) and to ACh(ACh 10 μmol/L: P = 0.001; ACh 100 μmol/L: P = 0.001) elicited by CORM-3 alone. L-NNA co-administered with granisetron and CORM-3 abolished the potentiating effect of CORM-3 on granisetron on both the EFSinduced and ACh-induced contractile response.CONCLUSION Taken together, findings from in vivo and in vitro studies suggest that the HO/CO pathway is involved in the constipating effects of granisetron.     

Subcortical damage and cortical functional changes in men and women with Fabry disease: a multifaceted MR study

PURPOSE: To prospectively compare brain magnetic resonance (MR) imaging and hydrogen 1 (1H) MR spectroscopy findings and to use functional MR imaging to explore the patterns of brain activation in men and women with Fabry disease (FD). MATERIALS AND METHODS: Eight men and eight women with FD (mean age, 38.8 years +/- 13.9 [standard deviation]) with absent or mild neurologic deficit and 16 healthy control subjects (eight men and eight women; mean age, 42.7 years +/- 15.3) gave informed consent to participate in the study, which was approved by the local ethical committee. Patients and control subjects underwent MR imaging, 1H MR spectroscopy of the frontal cortex and subcortical white matter, and functional MR imaging during repetitive flexion-extension of the last four fingers of the right hand. Extent of cerebral white matter damage was rated on fluid-attenuated inversion recovery MR images by using a visual score. Areas of activation were identified by using statistical parametric mapping software and the adoption of a height threshold of P < .001 (uncorrected) and an extent threshold of P < .05 (corrected). RESULTS: Men and women with FD showed a similar distribution of cerebral white matter changes, lacunar and cortical infarcts, small hemorrhages, and vertebrobasilar dolichoectasia. No significant (P > .05) difference was observed between patients with FD and control subjects for concentration of N-acetylaspartate, creatine, and choline. During the motor task, patients showed recruitment of additional cortical areas in comparison with control subjects. Increased activation of the contralateral sensorimotor area correlated (P = .002) with extent of white matter damage. CONCLUSION: Subcortical ischemic changes in men and women with FD are similar and are associated with increased recruitment of the sensorimotor network during a simple motor task, which might limit the functional effect of the white matter small-vessel disease.     

Three-Dimensional Co-Culture System of Human Osteoblasts and Osteoclast Precursors from Osteoporotic Patients as an Innovative Model to Study the Role of Nutrients: Focus on Vitamin K2

Several natural compounds, such as vitamin K2, have been highlighted for their positive effects on bone metabolism. It has been proposed that skeletal disorders, such as osteoporosis, may benefit from vitamin K2-based therapies or its regular intake. However, further studies are needed to better clarify the effects of vitamin K2 in bone disorders. To this aim, we developed in vitro a three-dimensional (3D) cell culture system one step closer to the bone microenvironment based on co-culturing osteoblasts and osteoclasts precursors obtained from bone specimens and peripheral blood of the same osteoporotic patient, respectively. Such a 3-D co-culture system was more informative than the traditional 2-D cell cultures when responsiveness to vitamin K2 was analyzed, paving the way for data interpretation on single patients. Following this approach, the anabolic effects of vitamin K2 on the osteoblast counterpart were found to be correlated with bone turnover markers measured in osteoporotic patients’ sera. Overall, our data suggest that co-cultured osteoblasts and osteoclast precursors from the same osteoporotic patient may be suitable to generate an in vitro 3-D experimental model that potentially reflects the individual’s bone metabolism and may be useful to predict personal responsiveness to nutraceutical or drug molecules designed to positively affect bone health.